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Cell_Biology_Alberts. Figure 6–1 The pathway from DNA to protein. The flow of genetic information from DNA to RNA (transcription) and from RNA to protein (translation) occurs in all living cells. human X chromosome: 155 million nucleotide base pairs (~5% of genome) total length of this section = 1.25 million nucleotide pairs


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Cell_Biology_Alberts. Figure 6–2 Schematic depiction of a small portion of the human X chromosome. As summarized in the key, the known protein-coding genes (starting with Abcd1 and ending with F8) are shown in dark gray, with coding regions (exons) indicated by bars that extend above and below the central line. Noncoding RNAs with known functions are indicated by purple diamonds. Yellow triangles indicate positions within protein-coding regions where the Neanderthal genome sequences codes for a different amino acid than the human genome. The stretch of yellow triangles in the Txtl1 gene appear to have been positively selected for since the divergence of Homo sapiens from Neanderthals some 200,000 years ago. Note that most of the proteins are identical between us and our extinct relative. The blue histogram indicates the extent to which portions of the human genome are conserved with other vertebrate species. It is likely that additional genes, currently unrecognized, also lie within this portion of the


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Cell_Biology_Alberts. indicates the extent to which portions of the human genome are conserved with other vertebrate species. It is likely that additional genes, currently unrecognized, also lie within this portion of the human genome.


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Cell_Biology_Alberts. Genes whose mutation causes an inherited human condition are indicated by red brackets. The Abcd1 gene codes for a protein that imports fatty acids into the peroxisome; mutations in the gene cause demylination of nerves which can result in cognition and movement disorders. Incontinentia pigmenti is a disease of the skin, hair, nails, teeth, and eyes. Hemophilia A is a bleeding disorder caused by mutations in the Factor VIII gene, which codes for a blood-clotting protein. Because males have only a single copy of the X chromosome, most of the conditions shown here affect only males; females that inherit one of these defective genes are often asymptomatic because a functional protein is made from their other X chromosome. (Courtesy of Alex Williams, obtained from the University of California, Genome Browser, http://genome.ucsc.edu) organism. Yet the cells in our body do this automatically, thousands of times a second.


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Cell_Biology_Alberts. The problems that cells face in decoding genomes can be appreciated by considering a tiny portion of the human genome (Figure 6–2). The region illustrated represents less than 1/2000th of our genome and includes at least 48 genes that encode proteins and 6 genes for noncoding RNAs. When we consider the entire human genome, we can only marvel at the capacity of our cells to rapidly and accurately handle such large amounts of information.


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Cell_Biology_Alberts. In this chapter, we explain how cells decode and use the information in their genomes. Much has been learned about how the genetic instructions written in an alphabet of just four “letters”—the four different nucleotides in DNA—direct the formation of a bacterium, a fruit fly, or a human. Nevertheless, we still have a great deal to discover about how the information stored in an organism’s genome produces even the simplest unicellular bacterium with 500 genes, let alone how it directs the development of a human with approximately 30,000 genes. An enormous amount of ignorance remains; many fascinating challenges therefore await the next generation of cell biologists.


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Cell_Biology_Alberts. Transcription and translation are the means by which cells read out, or express, the genetic instructions in their genes. Because many identical RNA copies can be made from the same gene, and each RNA molecule can direct the synthesis of many identical protein molecules, cells can synthesize a large amount of protein from a gene when necessary. But genes can be transcribed and translated with different efficiencies, allowing the cell to make vast quantities of some proteins and tiny amounts of others (Figure 6–3). Moreover, as we see in the next chapter, Figure 6–3 Genes can be expressed with different efficiencies. In this example, gene A is transcribed much more efficiently than gene B and each RNA molecule that it produces is also translated more frequently. This causes the amount of protein A in the cell to be much greater than that of protein B.


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Cell_Biology_Alberts. a cell can change (or regulate) the expression of each of its genes according to its needs—most commonly by controlling the production of its RNA. The first step a cell takes in reading out a needed part of its genetic instructions is to copy a particular portion of its DNA nucleotide sequence—a gene—into an RNA nucleotide sequence (Figure 6–4). The information in RNA, although copied into another chemical form, is still written in essentially the same language as it is in DNA—the language of a nucleotide sequence. Hence the name given to producing RNA molecules on DNA is transcription.


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Cell_Biology_Alberts. Like DNA, RNA is a linear polymer made of four different types of nucleotide subunits linked together by phosphodiester bonds (see Figure 6–4). It differs from DNA chemically in two respects: (1) the nucleotides in RNA are ribonucleotides— that is, they contain the sugar ribose (hence the name ribonucleic acid) rather than deoxyribose; (2) although, like DNA, RNA contains the bases adenine (A), guanine (G), and cytosine (C), it contains the base uracil (U) instead of the thymine (T) in DNA (Figure 6–5). Since U, like T, can base-pair by hydrogen-bonding with A (Figure 6–6), the complementary base-pairing properties described for DNA in Chapters 4 and 5 apply also to RNA (in RNA, G pairs with C, and A pairs with U). We also find other types of base pairs in RNA: for example, G occasionally pairs with U.


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Cell_Biology_Alberts. Although these chemical differences are slight, DNA and RNA differ quite dramatically in overall structure. Whereas DNA always occurs in cells as a double-stranded helix, RNA is single-stranded. An RNA chain can therefore fold up into a particular shape, just as a polypeptide chain folds up to form the final shape of a protein (Figure 6–7). As we see later in this chapter, the ability to fold into complex three-dimensional shapes allows some RNA molecules to have precise structural and catalytic functions. Transcription Produces RNA Complementary to One Strand of DNA


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Cell_Biology_Alberts. Transcription Produces RNA Complementary to One Strand of DNA The RNA in a cell is made by DNA transcription, a process that has certain similarities to the process of DNA replication discussed in Chapter 5. Transcription begins with the opening and unwinding of a small portion of the DNA double helix to expose the bases on each DNA strand. One of the two strands of the DNA double helix then acts as a template for the synthesis of an RNA molecule. As in DNA replication, the nucleotide sequence of the RNA chain is determined by the complementary base-pairing between incoming nucleotides and the DNA Figure 6–4 A short length of RNA. The phosphodiester chemical linkage between nucleotides in RNA is the same as that in DNA.


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Cell_Biology_Alberts. Figure 6–4 A short length of RNA. The phosphodiester chemical linkage between nucleotides in RNA is the same as that in DNA. Figure 6–5 The chemical structure of RNA. (A) RNA contains the sugar ribose, which differs from deoxyribose, the sugar used in DNA, by the presence of an additional –OH group. (B) RNA contains the base uracil, which differs from thymine, the equivalent base in DNA, by the absence of a –CH3 group. Figure 6–6 Uracil forms base pairs with adenine. The absence of a methyl group in U has no effect on base-pairing; thus, U-A base pairs closely resemble T-A base pairs (see Figure 4–4).


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Cell_Biology_Alberts. Figure 6–6 Uracil forms base pairs with adenine. The absence of a methyl group in U has no effect on base-pairing; thus, U-A base pairs closely resemble T-A base pairs (see Figure 4–4). template. When a good match is made (A with T, U with A, G with C, and C with G), the incoming ribonucleotide is covalently linked to the growing RNA chain in an enzymatically catalyzed reaction. The RNA chain produced by transcription— the transcript—is therefore elongated one nucleotide at a time, and it has a nucleotide sequence that is exactly complementary to the strand of DNA used as the template (Figure 6–8).


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Cell_Biology_Alberts. Transcription, however, differs from DNA replication in several crucial ways. Unlike a newly formed DNA strand, the RNA strand does not remain hydrogen-bonded to the DNA template strand. Instead, just behind the region where the ribonucleotides are being added, the RNA chain is displaced and the DNA helix re-forms. Thus, the RNA molecules produced by transcription are released from the DNA template as single strands. In addition, because they are copied from only a limited region of the DNA, RNA molecules are much shorter than DNA molecules. A DNA molecule in a human chromosome can be up to 250 million nucleotide-pairs long; in contrast, most RNAs are no more than a few thousand nucleotides long, and many are considerably shorter.


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Cell_Biology_Alberts. The enzymes that perform transcription are called RNA polymerases. Like the DNA polymerase that catalyzes DNA replication (discussed in Chapter 5), RNA polymerases catalyze the formation of the phosphodiester bonds that link the nucleotides together to form a linear chain. The RNA polymerase moves stepwise along the DNA, unwinding the DNA helix just ahead of the active site for polymerization to expose a new region of the template strand for complementary Figure 6–7 RNA can fold into specific structures. RNA is largely single-stranded, but it often contains short stretches of nucleotides that can form conventional base pairs with complementary sequences found elsewhere on the same molecule. These interactions, along with additional “nonconventional” base-pair interactions, allow an RNA molecule to fold into a three-dimensional structure that is determined by its sequence of nucleotides (Movie 6.1).


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Cell_Biology_Alberts. Diagram of a folded RNA structure showing only conventional base-pair interactions. (B) Structure with both conventional (red) and nonconventional (green) base-pair interactions. Structure of an actual RNA, one that catalyzes its own splicing (see p. 324). Each conventional base-pair interaction is indicated by a “rung” in the double helix. Bases in other configurations are indicated by broken rungs. base-pairing. In this way, the growing RNA chain is extended by one nucleotide at a time in the 5ʹ-to-3ʹdirection (Figure 6–9). The substrates are ribonucleoside triphosphates (ATP, CTP, UTP, and GTP); as in DNA replication, the hydrolysis of high-energy bonds provides the energy needed to drive the reaction forward (see Figure 5–4 and Movie 6.2).


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Cell_Biology_Alberts. The almost immediate release of the RNA strand from the DNA as it is synthesized means that many RNA copies can be made from the same gene in a relatively short time, with the synthesis of additional RNA molecules being started before the previous RNA molecules are completed (Figure 6–10). When RNA polymerase molecules follow hard on each other’s heels in this way, each moving at about 50 nucleotides per second, over a thousand transcripts can be synthesized in an hour from a single gene.


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Cell_Biology_Alberts. Although RNA polymerase catalyzes essentially the same chemical reaction as DNA polymerase, there are some important differences between the activities of the two enzymes. First, and most obviously, RNA polymerase catalyzes the linkage of ribonucleotides, not deoxyribonucleotides. Second, unlike the DNA polymerases involved in DNA replication, RNA polymerases can start an RNA chain without a primer. This difference is thought possible because transcription need not be as accurate as DNA replication (see Table 5–1, p. 244). RNA polymerases make about one mistake for every 104 nucleotides copied into RNA (compared with an error rate for direct copying by DNA polymerase of about one in 107 nucleotides); and the consequences of an error in RNA transcription are much less significant as RNA does not permanently store genetic information in cells. Finally, unlike DNA polymerases, which make their products in segments that are later stitched together, RNA polymerases are absolutely


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Cell_Biology_Alberts. as RNA does not permanently store genetic information in cells. Finally, unlike DNA polymerases, which make their products in segments that are later stitched together, RNA polymerases are absolutely processive; that is, the same RNA polymerase that begins an RNA molecule must finish it without dissociating from the DNA template.


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Cell_Biology_Alberts. Although not nearly as accurate as the DNA polymerases that replicate DNA, RNA polymerases nonetheless have a modest proofreading mechanism. If an incorrect ribonucleotide is added to the growing RNA chain, the polymerase can back up, and the active site of the enzyme can perform an excision reaction that resembles the reverse of the polymerization reaction, except that a water molecule replaces the pyrophosphate and a nucleoside monophosphate is released. Given that DNA and RNA polymerases both carry out template-dependent nucleotide polymerization, it might be expected that the two types of enzymes would be structurally related. However, x-ray crystallographic studies reveal that, other than containing a critical Mg2+ ion at the catalytic site, the two enzymes are quite different. Template-dependent nucleotide-polymerizing enzymes seem to have arisen at least twice during the early evolution of cells. One lineage led to the short region of DNA/RNA helix 5˜5˜ direction of


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Cell_Biology_Alberts. Figure 6–8 DNA transcription produces a single-stranded RNA molecule that is complementary to one strand of the DNA double helix. Note that the sequence of bases in the RNA molecule produced is the same as the sequence of bases in the non-template DNA strand, except that a U replaces every T base in the DNA.


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Cell_Biology_Alberts. Figure 6–9 DNA is transcribed by the enzyme RNA polymerase. The RNA polymerase (pale blue) moves stepwise along the DNA, unwinding the DNA helix at its active site indicated by the Mg2+ (red), which is required for catalysis. As it progresses, the polymerase adds nucleotides one by one to the RNA chain at the polymerization site, using an exposed DNA strand as a template. The RNA transcript is thus a complementary copy of one of the two DNA strands. A short region of DNA/RNA helix (approximately nine nucleotide pairs in length) is formed only transiently, and a “window” of DNA/RNA helix therefore moves along the DNA with the polymerase as the DNA double helix reforms behind it. The incoming nucleotides are in the form of ribonucleoside triphosphates (ATP, UTP, CTP, and GTP), and the energy stored in their phosphate– phosphate bonds provides the driving force for the polymerization reaction (see Figure 5–4). The figure, based on an x-ray crystallographic structure, shows a cutaway view


Cell_Biology_Alberts

Cell_Biology_Alberts. stored in their phosphate– phosphate bonds provides the driving force for the polymerization reaction (see Figure 5–4). The figure, based on an x-ray crystallographic structure, shows a cutaway view of the polymerase: the part facing the viewer has been sliced away to reveal the interior (Movie 6.3). (Adapted from P. Cramer et al., Science 288:640–649, 2000; PDB code: 1HQM.) modern DNA polymerases and reverse transcriptases discussed in Chapter 5, as well as to a few RNA polymerases from viruses. The other lineage formed all of the modern RNA polymerases that we discuss in this chapter.


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Cell_Biology_Alberts. Cells Produce Different Categories of RNA Molecules


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Cell_Biology_Alberts. The majority of genes carried in a cell’s DNA specify the amino acid sequence of proteins; the RNA molecules that are copied from these genes (which ultimately direct the synthesis of proteins) are called messenger RNA (mRNA) molecules. The final product of other genes, however, is the RNA molecule itself. These RNAs are known as noncoding RNAs because they do not code for protein. In a well-studied, single-celled eukaryote, the yeast Saccharomyces cerevisiae, over 1200 genes (more than 15% of the total) produce RNA as their final product. Humans may produce on the order of ten thousand noncoding RNAs. These RNAs, like proteins, serve as enzymatic, structural, and regulatory components for a wide variety of processes in the cell. In Chapter 5, we encountered one of them as the template carried by the enzyme telomerase. Although many of the noncoding RNAs are still mysterious, we shall see in this chapter that small nuclear RNA (snRNA) molecules direct the splicing of pre-mRNA to form


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Cell_Biology_Alberts. by the enzyme telomerase. Although many of the noncoding RNAs are still mysterious, we shall see in this chapter that small nuclear RNA (snRNA) molecules direct the splicing of pre-mRNA to form mRNA, that ribosomal RNA (rRNA) molecules form the core of ribosomes, and that transfer RNA (tRNA) molecules form the adaptors that select amino acids and hold them in place on a ribosome for incorporation into protein. In Chapter 7, we shall see that microRNA (miRNA) molecules and small interfering RNA (siRNA) molecules serve as key regulators of eukaryotic gene expression, and that piwi-interacting RNAs (piRNAs) protect animal germ lines from transposons; we also discuss the long noncoding RNAs (lncRNAs), a diverse set of RNAs whose functions are just being discovered (Table 6–1).


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Cell_Biology_Alberts. Figure 6–10 Transcription of two genes as observed under the electron microscope. The micrograph shows many molecules of RNA polymerase simultaneously transcribing each of two adjacent genes. Molecules of RNA polymerase are visible as a series of dots along the DNA with the newly synthesized transcripts (fine threads) attached to them. The RNA molecules (ribosomal RNAs) shown in this example are not translated into protein but are instead used directly as components of ribosomes, the machines on which translation takes place. The particles at the 5ʹ end (the free end) of each rRNA transcript are believed to reflect the beginnings of ribosome assembly. From the relative lengths of the newly synthesized transcripts, it can be deduced that the RNA polymerase molecules are transcribing from left to right. (Courtesy of Ulrich Scheer.)


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Cell_Biology_Alberts. Each transcribed segment of DNA is called a transcription unit. In eukaryotes, a transcription unit typically carries the information of just one gene, and therefore codes for either a single RNA molecule or a single protein (or group of related proteins if the initial RNA transcript is spliced in more than one way to produce different mRNAs). In bacteria, a set of adjacent genes is often transcribed as a unit; the resulting mRNA molecule therefore carries the information for several distinct proteins. Overall, RNA makes up a few percent of a cell’s dry weight, whereas proteins comprise about 50%. Most of the RNA in cells is rRNA; mRNA comprises only 3–5% of the total RNA in a typical mammalian cell. The mRNA population is made up of tens of thousands of different species, and there are on average only 10–15 molecules of each species of mRNA present in each cell. Signals Encoded in DNA Tell RNA Polymerase Where to Start and Stop


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Cell_Biology_Alberts. Signals Encoded in DNA Tell RNA Polymerase Where to Start and Stop To transcribe a gene accurately, RNA polymerase must recognize where on the genome to start and where to finish. The way in which RNA polymerases perform these tasks differs somewhat between bacteria and eukaryotes. Because the processes in bacteria are simpler, we discuss them first.


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Cell_Biology_Alberts. The initiation of transcription is an especially important step in gene expression because it is the main point at which the cell regulates which proteins are to be produced and at what rate. The bacterial RNA polymerase core enzyme is a multisubunit complex that synthesizes RNA using the DNA template as a guide. An additional subunit called sigma (σ) factor associates with the core enzyme and assists it in reading the signals in the DNA that tell it where to begin transcribing (Figure 6–11). Together, σ factor and core enzyme are known as the RNA polymerase holoenzyme; this complex adheres only weakly to bacterial DNA when the two collide, and a holoenzyme typically slides rapidly along the long DNA molecule and then dissociates. However, when the polymerase holoenzyme slides into a special sequence of nucleotides indicating the starting point for RNA synthesis called a promoter, the polymerase binds tightly, because its σ factor makes specific contacts with the edges of bases


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Cell_Biology_Alberts. a special sequence of nucleotides indicating the starting point for RNA synthesis called a promoter, the polymerase binds tightly, because its σ factor makes specific contacts with the edges of bases exposed on the outside of the DNA double helix (step 1 in Figure 6–11A).


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Cell_Biology_Alberts. The tightly bound RNA polymerase holoenzyme at a promoter opens up the double helix to expose a short stretch of nucleotides on each strand (step 2 in Figure 6–11A). The region of unpaired DNA (about 10 nucleotides) is called the transcription bubble and it is stabilized by the binding of σ factor to the unpaired bases on one of the exposed strands. The other exposed DNA strand then acts as a template for complementary base-pairing with incoming ribonucleotides, two of which are joined together by the polymerase to begin an RNA chain (step 3 in Figure 6–11A). The first ten or so nucleotides of RNA are synthesized using a “scrunching” mechanism, in which RNA polymerase remains bound to the promoter and pulls the upstream DNA into its active site, thereby expanding the transcription bubble. This process creates considerable stress and the short RNAs are often released, thereby relieving the stress and forcing the polymerase, which remains in place, to begin synthesis over again.


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Cell_Biology_Alberts. bubble. This process creates considerable stress and the short RNAs are often released, thereby relieving the stress and forcing the polymerase, which remains in place, to begin synthesis over again. Eventually this process of abortive initiation is overcome and the stress generated by scrunching helps the core enzyme to break free of its interactions with the promoter DNA (step 4 in Figure 6–11A) and discard the σ factor (step 5 in Figure 6–11A). At this point, the polymerase begins to move down the DNA, synthesizing RNA, in a stepwise fashion: the polymerase moves forward one base pair for every nucleotide added. During this process, the transcription bubble continually expands at the front of the polymerase and contracts at its rear. Chain elongation continues (at a speed of approximately 50 nucleotides/sec for bacterial RNA polymerases) until the enzyme encounters a second signal, the terminator (step 6 in Figure 6–11A), where the polymerase halts and releases both the newly made


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Cell_Biology_Alberts. 50 nucleotides/sec for bacterial RNA polymerases) until the enzyme encounters a second signal, the terminator (step 6 in Figure 6–11A), where the polymerase halts and releases both the newly made RNA molecule and the DNA template (step 7 in Figure 6–11A). The free polymerase core enzyme then reassociates with a free σ factor to form a holoenzyme that can begin the process of transcription again (step 8 in Figure 6–11A).


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Cell_Biology_Alberts. The process of transcription initiation is complicated and requires that the RNA polymerase holoenzyme and the DNA undergo a series of conformational changes. We can view these changes as opening up and positioning the DNA in the active site followed by a successive tightening of the enzyme around the DNA and RNA to ensure that it does not dissociate before it has finished transcribing a gene. If an RNA polymerase does dissociate prematurely, it must start over again at the promoter.


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Cell_Biology_Alberts. How do the termination signals in the DNA stop the elongating polymerase? For most bacterial genes, a termination signal consists of a string of A-T nucleotide pairs preceded by a twofold symmetric DNA sequence, which, when transcribed into RNA, folds into a “hairpin” structure through Watson–Crick base-pairing (see Figure 6–11A). As the polymerase transcribes across a terminator, the formation of the hairpin helps to disengage the RNA transcript from the active site (step 7 in Figure 6–11A). The process of termination provides an example of a common theme in this chapter: the folding of RNA into specific structures affects many steps in decoding the genome.


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Cell_Biology_Alberts. As we have just seen, the processes of transcription initiation and termination involve a complicated series of structural transitions in protein, DNA, and RNA molecules. The signals encoded in DNA that specify these transitions are often difficult for researchers to recognize. Indeed, a comparison of many different bacterial promoters reveals a surprising degree of variation. Nevertheless, they all contain related sequences, reflecting aspects of the DNA that are recognized directly


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Cell_Biology_Alberts. Figure 6–11 The transcription cycle of bacterial RNA polymerase. (A) In step 1, the RNA polymerase holoenzyme (polymerase core enzyme plus σ factor) assembles and then locates a promoter DNA sequence (see Figure 6–12). The polymerase opens (unwinds) the DNA at the position at which transcription is to begin (step 2) and begins transcribing (step 3). This initial RNA synthesis (abortive initiation) is relatively inefficient as short, unproductive transcripts are often released. However, once RNA polymerase has managed to synthesize about 10 nucleotides of RNA, it breaks its interactions with the promoter DNA (step 4) and eventually releases σ factor—as the polymerase tightens around the DNA and shifts to the elongation mode of RNA synthesis, moving along the DNA (step 5). During the elongation mode, transcription is highly processive, with the polymerase leaving the DNA template and releasing the newly transcribed RNA only when it encounters a termination signal (steps 6 and 7).


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Cell_Biology_Alberts. elongation mode, transcription is highly processive, with the polymerase leaving the DNA template and releasing the newly transcribed RNA only when it encounters a termination signal (steps 6 and 7). Termination signals are typically encoded in DNA, and many function by forming an RNA hairpin-like structure that destabilizes the polymerase’s hold on the RNA.


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Cell_Biology_Alberts. In bacteria, all RNA molecules are synthesized by a single type of RNA polymerase, and the cycle depicted in the figure therefore applies to the production of mRNAs as well as structural and catalytic RNAs. (B) Two-dimensional image of an elongating bacterial RNA polymerase, as determined by atomic force microscopy (see Figure 9–33). (C) Interpretation of the image in (B). (Adapted from K.M. Herbert et al., Annu. Rev. Biochem. 77:149–176, 2008.) frequency of nucleotide in each position (%) sequence and sequence logo for the major class of E. coli promoters.


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Cell_Biology_Alberts. (A) On the basis of a comparison of 300 promoters, the frequencies of each of the four nucleotides at each position in the promoter are given. The consensus sequence, shown below the graph, reflects the most common nucleotide found at each position in the collection of promoters. These promoters are characterized by two hexameric DNA sequences—the –35 sequence and the –10 sequence, named for their approximate location relative to the start point of transcription (designated +1). The sequence of nucleotides between the –35 and –10 hexamers shows no significant similarities among promoters.


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Cell_Biology_Alberts. For convenience, the nucleotide sequence of a single strand of DNA is shown; in reality, promoters are double-stranded by the σ factor. These common features are often summarized in the form of a consensus sequence (Figure 6–12). A consensus nucleotide sequence is derived by comparing many sequences with the same basic function and tallying up the most common nucleotides found at each position. It therefore serves as a summary or “average” of a large number of individual nucleotide sequences. A more accurate way of displaying the range of DNA sequences recognized by a protein is through the use of a sequence logo, which reveals the relative frequencies of each nucleotide at each position (Figure 6–12C).


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Cell_Biology_Alberts. The DNA sequences of individual bacterial promoters differ in ways that determine their strength (the number of initiation events per unit time of the promoter). Evolutionary processes have fine-tuned each to initiate as often as necessary and have thereby created a wide spectrum of promoter strengths. Promoters for genes that code for abundant proteins are much stronger than those associated with genes that encode rare proteins, and the nucleotide sequences of their promoters are responsible for these differences. Like bacterial promoters, transcription terminators also have a wide range of sequences, with the potential to form a simple hairpin RNA structure being the most important common feature. Since an almost unlimited number of nucleotide sequences have this potential, terminator sequences are even more heterogeneous than promoter sequences.


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Cell_Biology_Alberts. We have discussed bacterial promoters and terminators in some detail to illustrate an important point regarding the analysis of genome sequences. Although we know a great deal about bacterial promoters and terminators and can construct consensus sequences that summarize their most salient features, their variation in nucleotide sequence makes it difficult to definitively locate them simply DNA. The nucleotides shown in the figure are recognized by σ factor, a subunit of the RNA polymerase holoenzyme.


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Cell_Biology_Alberts. DNA. The nucleotides shown in the figure are recognized by σ factor, a subunit of the RNA polymerase holoenzyme. (B) The distribution of spacing between the –35 and –10 hexamers found in E. coli promoters. (C) A sequence logo displaying the same information as in panel (A). Here, the height of each letter is proportional to the frequency at which that base occurs at that position across a wide variety of promoter sequences. The total height of all the letters at each position is proportional to the information content (expressed in bits) at that position. For example, the total information content of a position that can tolerate several different bases is small (see the last three bases of the –35 sequences), but statistically greater than random.


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Cell_Biology_Alberts. DNA of E. coli chromosome gene a by analysis of the nucleotide sequence of a genome. It is even more difficult to locate analogous sequences in eukaryotic genomes, due in part to the excess DNA carried in these genomes. Often we need additional information, some of it from direct experimentation, to locate and accurately interpret the short DNA signals in genomes. As shown in Figure 6–11, promoter sequences are asymmetric, ensuring that RNA polymerase can bind in only one orientation. Because the polymerase can synthesize RNA only in the 5ʹ-to-3ʹ direction, the promoter orientation specifies the strand to be used as a template. Genome sequences reveal that the DNA strand that is used as the template for RNA synthesis varies from gene to gene, depending on the orientation of the promoter (Figure 6–13). Having considered transcription in bacteria, we now turn to the situation in eukaryotes, where the synthesis of RNA molecules is a much more elaborate affair.


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Cell_Biology_Alberts. Having considered transcription in bacteria, we now turn to the situation in eukaryotes, where the synthesis of RNA molecules is a much more elaborate affair. In contrast to bacteria, which contain a single type of RNA polymerase, eukaryotic nuclei have three: RNA polymerase I, RNA polymerase II, and RNA polymerase III. The three polymerases are structurally similar to one another and share some common subunits, but they transcribe different categories of genes (Table 6–2). RNA polymerases I and III transcribe the genes encoding transfer RNA, ribosomal RNA, and various small RNAs. RNA polymerase II transcribes most genes, including all those that encode proteins, and our subsequent discussion therefore focuses on this enzyme. Eukaryotic RNA polymerase II has many structural similarities to bacterial RNA polymerase (Figure 6–14). But there are several important differences in the way in which the bacterial and eukaryotic enzymes function, two of which concern us immediately. 1.


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Cell_Biology_Alberts. 1. While bacterial RNA polymerase requires only a single transcriptioninitiation factor (σ) to begin transcription, eukaryotic RNA polymerases require many such factors, collectively called the general transcription factors. 2. Eukaryotic transcription initiation must take place on DNA that is packaged into nucleosomes and higher-order forms of chromatin structure (described in Chapter 4), features that are absent from bacterial chromosomes.


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Cell_Biology_Alberts. Figure 6–13 Directions of transcription along a short portion of a bacterial chromosome. Some genes are transcribed using one DNA strand as a template, while others are transcribed using the other DNA strand. The direction of transcription is determined by the promoter at the beginning of each gene (green arrowheads). This diagram shows approximately 0.2% (9000 base pairs) of the E. coli chromosome. The genes transcribed from left to right use the bottom DNA strand as the template; those transcribed from right to left use the top strand as the template. RNA Polymerase II Requires a Set of General Transcription Factors


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Cell_Biology_Alberts. RNA Polymerase II Requires a Set of General Transcription Factors The general transcription factors help to position eukaryotic RNA polymerase correctly at the promoter, aid in pulling apart the two strands of DNA to allow transcription to begin, and release RNA polymerase from the promoter to start its elongation mode. The proteins are “general” because they are needed at nearly all promoters used by RNA polymerase II. They consist of a set of interacting proteins denoted arbitrarily as TFIIA, TFIIB, TFIIC, TFIID, and so on (TFII standing for “transcription factor for polymerase II).” In a broad sense, the eukaryotic general transcription factors carry out functions equivalent to those of the σ factor in bacteria; indeed, portions of TFIIF have the same three-dimensional structure as the equivalent portions of σ.


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Cell_Biology_Alberts. Figure 6–15 illustrates how the general transcription factors assemble at promoters used by RNA polymerase II, and Table 6–3 summarizes their activities. The assembly process begins when TFIID binds to a short double-helical DNA sequence primarily composed of T and A nucleotides. For this reason, this sequence is known as the TATA sequence, or TATA box, and the subunit of TFIID that recognizes it is called TBP (for TATA-binding protein). The TATA box is typically located 25 nucleotides upstream from the transcription start site. It is not the only DNA sequence that signals the start of transcription (Figure 6–16), but for most polymerase II promoters it is the most important. The binding of TFIID


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Cell_Biology_Alberts. Figure 6–15 Initiation of transcription of a eukaryotic gene by RNA polymerase II. To begin transcription, RNA polymerase requires several general transcription factors. (A) The promoter contains a DNA sequence called the TATA box, which is located 25 nucleotides away from the site at which transcription is initiated. (B) Through its subunit TBP, TFIID recognizes and binds the TATA box, which then enables the adjacent binding of TFIIB (C). For simplicity the DNA distortion produced by the binding of TFIID (see Figure 6–17) is not shown. (D) The rest of the general transcription factors, as well as the RNA polymerase itself, assemble at the promoter. (E) TFIIH then uses energy from ATP hydrolysis to pry apart the DNA double helix at the transcription start point, locally exposing the template strand. TFIIH also phosphorylates RNA polymerase II, changing its conformation so that the polymerase is released from the general factors and can begin the elongation phase of transcription. As


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Cell_Biology_Alberts. strand. TFIIH also phosphorylates RNA polymerase II, changing its conformation so that the polymerase is released from the general factors and can begin the elongation phase of transcription. As shown, the site of phosphorylation is a long C-terminal polypeptide tail, also called the C-terminal domain (CTD), that extends from the polymerase molecule. The assembly scheme shown in the figure was deduced from experiments performed in vitro, and the exact order in which the general transcription factors assemble on promoters probably varies from gene to gene in vivo. The general transcription factors are highly conserved; some of those from human cells can be replaced in biochemical experiments by the corresponding factors from simple yeasts.


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Cell_Biology_Alberts. Figure 6–14 Structural similarity between a bacterial RNA polymerase and a eukaryotic RNA polymerase II. Regions of the two RNA polymerases that have similar structures are indicated in green. The eukaryotic polymerase is larger than the bacterial enzyme (12 subunits instead of 5), and some of the additional regions are shown in gray. The blue spheres represent Zn atoms that serve as structural components of the polymerases, and the red sphere represents the Mg atom present at the active site, where polymerization takes place. The RNA polymerases in all modern-day cells (bacteria, archaea, and eukaryotes) are closely related, indicating that the basic features of the enzyme were in place before the divergence of the three major branches of life. (Courtesy of


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Cell_Biology_Alberts. P. Cramer and R. Kornberg.) start of transcription TATA box (D) UTP, ATP CTP, GTP FACTOR RELEASE causes a large distortion in the DNA of the TATA box (Figure 6–17). This distortion is thought to serve as a physical landmark for the location of an active promoter in the midst of a very large genome, and it brings DNA sequences on both sides of the distortion closer together to allow for subsequent protein assembly steps. Other factors then assemble, along with RNA polymerase II, to form a complete transcription initiation complex (see Figure 6–15). The most complicated of the general transcription factors is TFIIH. Consisting of nine subunits, it is nearly as large as RNA polymerase II itself and, as we shall see shortly, performs several enzymatic steps needed for the initiation of transcription.


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Cell_Biology_Alberts. After forming a transcription initiation complex on the promoter DNA, RNA polymerase II must gain access to the template strand at the transcription start point. TFIIH, which contains a DNA helicase as one of its subunits, makes this step possible by hydrolyzing ATP and unwinding the DNA, thereby exposing the template strand. Next, RNA polymerase II, like the bacterial polymerase, remains at the promoter synthesizing short lengths of RNA until it undergoes a series of conformational changes that allow it to move away from the promoter and enter the elongation phase of transcription. A key step in this transition is the addition of phosphate groups to the “tail” of the RNA polymerase (known as the CTD or C-terminal domain). In humans, the CTD consists of 52 tandem repeats of a subunit of TFIID C/T C/T A N T/A C/T C/T TFIID


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Cell_Biology_Alberts. Figure 6–16 Consensus sequences found in the vicinity of eukaryotic RNA polymerase II start points. The name given to each consensus sequence (first column) and the general transcription factor that recognizes it (last column) are indicated. N indicates any nucleotide, and two nucleotides separated by a slash indicate an equal probability of either nucleotide at the indicated position. In reality, each consensus sequence is a shorthand representation of a histogram similar to that of Figure 6–12. For most RNA polymerase II transcription start points, only two or three of the four sequences are present. For example, many polymerase II promoters have a TATA box sequence, but those that do not typically have a “strong” INR sequence. Although most of the DNA sequences that influence transcription initiation are located upstream of the transcription start point, a few, such as the DPE shown in the figure, are located in the transcribed region.


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Cell_Biology_Alberts. seven-amino-acid sequence, which extend from the RNA polymerase core structure. During transcription initiation, the serine located at the fifth position in the repeat sequence (Ser5) is phosphorylated by TFIIH, which contains a protein kinase in one of its subunits (see Figure 6–15D and E). The polymerase can then disengage from the cluster of general transcription factors. During this process, it undergoes a series of conformational changes that tighten its interaction with DNA, and it acquires new proteins that allow it to transcribe for long distances, in some cases for many hours, without dissociating from DNA.


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Cell_Biology_Alberts. Once the polymerase II has begun elongating the RNA transcript, most of the general transcription factors are released from the DNA so that they are available to initiate another round of transcription with a new RNA polymerase molecule. As we see shortly, the phosphorylation of the tail of RNA polymerase II has an additional function: it causes components of the RNA-processing machinery to load onto the polymerase and thus be positioned to modify the newly transcribed RNA as it emerges from the polymerase. Polymerase II Also Requires Activator, Mediator, and Chromatin-Modifying Proteins


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Cell_Biology_Alberts. Studies of RNA polymerase II and its general transcription factors acting on DNA templates in purified in vitro systems established the model for transcription initiation just described. However, as discussed in Chapter 4, DNA in eukaryotic cells is packaged into nucleosomes, which are further arranged in higher-order chromatin structures. As a result, transcription initiation in a eukaryotic cell is more complex and requires more proteins than it does on purified DNA. First, gene regulatory proteins known as transcriptional activators must bind to specific sequences in DNA (called enhancers) and help to attract RNA polymerase II to the start point of transcription (Figure 6–18). We discuss the role of these activators in Chapter 7, because they are one of the main ways in which cells regulate expression of their genes. Here we simply note that their presence on DNA is required for transcription initiation in a eukaryotic cell. Second, eukaryotic transcription initiation in vivo


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Cell_Biology_Alberts. regulate expression of their genes. Here we simply note that their presence on DNA is required for transcription initiation in a eukaryotic cell. Second, eukaryotic transcription initiation in vivo requires the presence of a large protein complex known as Mediator, which allows the activator proteins to communicate properly with the polymerase II and with the general transcription factors. Finally, transcription initiation in a eukaryotic cell typically requires the recruitment of chromatin-modifying enzymes, including chromatin remodeling complexes and


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Cell_Biology_Alberts. Figure 6–17 Three-dimensional structure of TbP (TATA-binding protein) bound to DNA. The TBP is the subunit of the general transcription factor TFIID that is responsible for recognizing and binding to the TATA box sequence in the DNA (red). The unique DNA bending caused by TBP—kinks in the double helix separated by partly unwound DNA—is thought to serve as a landmark that helps to attract the other general transcription factors (Movie 6.4). TBP is a single polypeptide chain that is folded into two very similar domains (blue and green). (Adapted from J.L. Kim et al., Nature 365:520–527, 1993. With permission from Macmillan Publishers Ltd.) start of (binding site for GENERAL TRANSCRIPTION FACTORS, RNA POLYMERASE, MEDIATOR, CHROMATIN REMODELING COMPLEXES, AND HISTONE-MODIFYING ENZYMES


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Cell_Biology_Alberts. GENERAL TRANSCRIPTION FACTORS, RNA POLYMERASE, MEDIATOR, CHROMATIN REMODELING COMPLEXES, AND HISTONE-MODIFYING ENZYMES RNA polymerase bound to histone-modifying enzymes. As discussed in Chapter 4, both types of enzymes can increase access to the DNA in chromatin, and by doing so they facilitate the assembly of the transcription initiation machinery onto DNA. As illustrated in Figure 6–18, many proteins (well over 100 individual subunits) must assemble at the start point of transcription to initiate transcription in a eukaryotic cell. The order of assembly of these proteins does not seem to follow a prescribed pathway; rather, the order differs from gene to gene. Indeed, some of these different protein complexes may be brought to DNA as preformed subassemblies.


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Cell_Biology_Alberts. To begin transcribing, RNA polymerase II must be released from this large complex of proteins. In addition to the steps described in Figure 6–14, this release often requires the in situ proteolysis of the activator protein. We shall return to some of these issues, including the role of chromatin remodeling complexes and histone-modifying enzymes, in Chapter 7, where we discuss how eukaryotic cells regulate the process of transcription initiation.


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Cell_Biology_Alberts. Once RNA polymerase has initiated transcription, it moves jerkily, pausing at some DNA sequences and rapidly transcribing through others. Elongating RNA polymerases, both bacterial and eukaryotic, are associated with a series of elongation factors, proteins that decrease the likelihood that RNA polymerase will dissociate before it reaches the end of a gene. These factors typically associate with RNA polymerase shortly after initiation and help the polymerase move through the wide variety of different DNA sequences that are found in genes. Eukaryotic RNA polymerases must also contend with chromatin structure as they move along a DNA template, and they are typically aided by ATP-dependent chromatin remodeling complexes that either move with the polymerase or may simply seek out and rescue the occasional stalled polymerase. In addition, histone chaperones help by partially disassembling nucleosomes in front of a moving RNA polymerase and assembling them behind.


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Cell_Biology_Alberts. As RNA polymerase moves along a gene, some of the enzymes bound to it modify the histones, leaving behind a record of where the polymerase has been. Although it is not clear exactly how the cell uses this information, it may aid in Figure 6–18 Transcription initiation by RNA polymerase II in a eukaryotic cell.


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Cell_Biology_Alberts. Transcription initiation in vivo requires the presence of transcription activator proteins. As described in Chapter 7, these proteins bind to specific short sequences in DNA. Although only one is shown here, a typical eukaryotic gene utilizes many transcription activator proteins, which in combination determine its rate and pattern of transcription. Sometimes acting from a distance of several thousand nucleotide pairs (indicated by the dashed DNA molecule), these proteins help RNA polymerase, the general transcription factors, and Mediator all to assemble at the promoter. In addition, activators attract ATP-dependent chromatin remodeling complexes and histone-modifying enzymes. One of the main roles of Mediator is to coordinate the assembly of all these proteins at the promoter so that transcription can begin. As discussed in Chapter 4, the “default” state of chromatin is a condensed fiber (see Figure 4–28), and this is likely to be the form of DNA upon which most transcription is


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Cell_Biology_Alberts. transcription can begin. As discussed in Chapter 4, the “default” state of chromatin is a condensed fiber (see Figure 4–28), and this is likely to be the form of DNA upon which most transcription is initiated. For simplicity, the chromatin is not shown in this figure.


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Cell_Biology_Alberts. transcribing a gene over and over again once it has become active for the first time. It may also be used to coordinate transcription elongation with the processing of RNA as it emerges from RNA polymerase, a topic we discuss later in this chapter.


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Cell_Biology_Alberts. There is yet another barrier to elongating RNA polymerases, both bacterial and eukaryotic, one that also applies to DNA polymerases, as discussed in Chapter 5 (see Figure 5–20). To describe this issue in more detail, we need first to consider a subtle property inherent in the DNA double helix called DNA supercoiling. DNA supercoiling is the name given to a conformation that DNA adopts in response to superhelical tension; alternatively, creating loops or coils in a double-helical DNA molecule can create such tension. Figure 6–19 illustrates why. There are approximately 10 nucleotide pairs for every helical turn in a DNA double helix. If we imagine a helix whose two ends are fixed with respect to each other (as they are in a DNA circle, such as a bacterial chromosome, or in a tightly clamped loop, as is thought to exist in eukaryotic chromosomes), one large DNA supercoil will form to compensate for each 10 nucleotide pairs that are opened (unwound). The formation of this supercoil is


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Cell_Biology_Alberts. loop, as is thought to exist in eukaryotic chromosomes), one large DNA supercoil will form to compensate for each 10 nucleotide pairs that are opened (unwound). The formation of this supercoil is energetically favorable because it restores a normal helical twist to the base-paired regions that remain, which would otherwise need to be overwound because of the fixed ends.


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Cell_Biology_Alberts. RNA polymerase creates superhelical tension as it moves along a stretch of DNA that is anchored at its ends (see Figure 6–19C). As long as the polymerase is not free to rotate rapidly (and such rotation is unlikely given the size of RNA polymerases and their attached transcripts), a moving polymerase generates positive superhelical tension in the DNA in front of it and negative helical tension behind it. For eukaryotes, this situation is thought to provide a bonus: although the positive superhelical tension ahead of the polymerase makes the DNA helix DNA with free end DNA with fxed ends Figure 6–19 Superhelical tension in DNA causes DNA supercoiling. (A) For a DNA molecule with one free end (or a nick in one strand that serves as a swivel), the DNA double helix rotates by one turn for every 10 nucleotide pairs opened.


Cell_Biology_Alberts

Cell_Biology_Alberts. (B) If rotation is prevented, superhelical tension is introduced into the DNA by helix opening. In the example shown, the DNA helix contains 10 helical turns, one of which is opened. One way of accommodating the tension created would be to increase the helical twist from 10 to 11 nucleotide pairs per turn in the double helix that remains. The DNA helix, however, resists such a deformation in a springlike fashion, preferring to relieve the superhelical tension by bending into supercoiled loops. As a result, one DNA supercoil forms in the DNA double helix for every 10 nucleotide pairs opened. The supercoil formed in this case is a positive supercoil. (C) Supercoiling of DNA is induced by a protein tracking through the DNA double helix. The two ends of the DNA shown here are unable to rotate freely relative to each other, and the protein molecule is assumed also to be prevented from rotating freely as it moves. Under these conditions, the movement of the protein causes an excess of


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Cell_Biology_Alberts. freely relative to each other, and the protein molecule is assumed also to be prevented from rotating freely as it moves. Under these conditions, the movement of the protein causes an excess of helical turns to accumulate in the DNA helix ahead of the protein and a deficit of helical turns to arise in the DNA behind the protein, as shown.


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Cell_Biology_Alberts. more difficult to open, the tension should facilitate the partial unwrapping of the DNA in nucleosomes, inasmuch as the release of DNA from the histone core helps to relax this tension.


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Cell_Biology_Alberts. Any protein that propels itself alone along a DNA strand of a double helix, such as a DNA helicase or an RNA polymerase, tends to generate superhelical tension. In eukaryotes, DNA topoisomerase enzymes rapidly remove this superhelical tension (see pp. 251–253). But in bacteria a specialized topoisomerase called DNA gyrase uses the energy of ATP hydrolysis to pump supercoils continuously into the DNA, thereby maintaining the DNA under constant tension. These are negative supercoils, having the opposite handedness from the positive supercoils that form when a region of DNA helix opens (see Figure 6–19B). Whenever a region of helix opens, it removes these negative supercoils from bacterial DNA, reducing the superhelical tension. DNA gyrase therefore makes the opening of the DNA helix in bacteria energetically favorable compared with helix opening in DNA that is not supercoiled. For this reason, it facilitates those genetic processes in bacteria, such as the initiation of transcription by


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Cell_Biology_Alberts. energetically favorable compared with helix opening in DNA that is not supercoiled. For this reason, it facilitates those genetic processes in bacteria, such as the initiation of transcription by bacterial RNA polymerase, that require helix opening (see Figure 6–11).


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Cell_Biology_Alberts. Transcription Elongation in Eukaryotes Is Tightly Coupled to RNA Processing We have seen that bacterial mRNAs are synthesized by the RNA polymerase starting and stopping at specific spots on the genome. The situation in eukaryotes is substantially different. In particular, transcription is only the first of several steps needed to produce a mature mRNA molecule. Other critical steps are the covalent modification of the ends of the RNA and the removal of intron sequences that are discarded from the middle of the RNA transcript by the process of RNA splicing (Figure 6–20). Both ends of eukaryotic mRNAs are modified: by capping on the 5ʹ end and by polyadenylation of the 3ʹ end (Figure 6–21). These special ends allow the cell to assess whether both ends of an mRNA molecule are present (and if the message is therefore intact) before it exports the RNA from the nucleus and translates it


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Cell_Biology_Alberts. Figure 6–20 Comparison of the steps leading from gene to protein in eukaryotes and bacteria. The final level of a protein in the cell depends on the efficiency of each step and on the rates of degradation of the RNA and protein molecules. (A) In eukaryotic cells, the mRNA molecule resulting from transcription contains both coding (exon) and noncoding (intron) sequences. Before it can be translated into protein, the two ends of the RNA are modified, the introns are removed by an enzymatically catalyzed RNA splicing reaction, and the resulting mRNA is transported from the nucleus to the cytoplasm. For convenience, the steps in this figure are depicted as occurring one at a time; in reality, many occur concurrently. For example, the RNA cap is added and splicing begins before transcription has been completed. Because of the coupling between transcription and RNA processing, intact primary transcripts—the full-length RNAs that would, in theory, be produced if no processing had


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Cell_Biology_Alberts. has been completed. Because of the coupling between transcription and RNA processing, intact primary transcripts—the full-length RNAs that would, in theory, be produced if no processing had occurred—are found only rarely. (B) In prokaryotes, the production of mRNA is much simpler. The 5ʹ end of an mRNA molecule is produced by the initiation of transcription, and the 3ʹ end is produced by the termination of transcription. Since prokaryotic cells lack a nucleus, transcription and translation take place in a common compartment, and the translation of a bacterial mRNA often begins before its synthesis has been completed.


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Cell_Biology_Alberts. prokaryotic mRNA 5˜ end of coding noncoding 7-methylguanosine primary transcript sequence sequence


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Cell_Biology_Alberts. Figure 6–21 A comparison of the structures of prokaryotic and eukaryotic mRNA molecules. (A) The 5ʹ and 3ʹ ends of a bacterial mRNA are the unmodified ends of the chain synthesized by the RNA polymerase, which initiates and terminates transcription at those points, respectively. The corresponding ends of a eukaryotic mRNA are formed by adding a 5ʹ cap and by cleavage of the pre-mRNA transcript near the 3ʹ end and the addition of a poly-A tail, respectively. The figure also illustrates another difference between the prokaryotic and eukaryotic mRNAs: bacterial mRNAs can contain the instructions for several different proteins, whereas eukaryotic mRNAs nearly always contain the information for only a single protein. (B) The structure of the cap at the 5ʹ end of eukaryotic mRNA molecules. Note the unusual 5ʹ-to-5ʹ linkage of the 7-methyl G to the remainder of the RNA. Many eukaryotic mRNAs carry an additional modification: methylation of the 2ʹ-hydroxyl group of the ribose sugar at the 5ʹ


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Cell_Biology_Alberts. the unusual 5ʹ-to-5ʹ linkage of the 7-methyl G to the remainder of the RNA. Many eukaryotic mRNAs carry an additional modification: methylation of the 2ʹ-hydroxyl group of the ribose sugar at the 5ʹ end of the primary transcript (see Figure 6–23).


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Cell_Biology_Alberts. into protein. RNA splicing joins together the different portions of a protein-coding sequence, and it provides eukaryotes with the ability to synthesize several different proteins from the same gene.


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Cell_Biology_Alberts. A simple strategy has evolved to couple all of the above RNA processing steps to transcription elongation. As discussed previously, a key step in transcription initiation by RNA polymerase II is the phosphorylation of the RNA polymerase II tail, also called the CTD (C-terminal domain). This phosphorylation, which proceeds gradually as the RNA polymerase initiates transcription and moves along the DNA, not only helps dissociate the RNA polymerase II from other proteins present at the start point of transcription, but also allows a new set of proteins to associate with the RNA polymerase tail that function in transcription elongation and RNA processing. As discussed next, some of these processing proteins are thought to “hop” from the polymerase tail onto the nascent RNA molecule to begin processing it as it emerges from the RNA polymerase. Thus, we can view RNA polymerase II in its elongation mode as an RNA factory that not only moves along the DNA synthesizing an RNA molecule, but


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Cell_Biology_Alberts. processing it as it emerges from the RNA polymerase. Thus, we can view RNA polymerase II in its elongation mode as an RNA factory that not only moves along the DNA synthesizing an RNA molecule, but also processes the RNA that it produces (Figure 6–22). Fully extended, the CTD is nearly 10 times longer than the remainder of RNA polymerase. As a flexible protein domain, it serves as a scaffold or tether, holding a variety of proteins close by so that they can rapidly act when needed. This strategy, which greatly speeds up the overall rate of a series of consecutive reactions, is one that is commonly utilized in the cell (see Figures 4–58 and 16–18).


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Cell_Biology_Alberts. RNA Capping Is the First Modification of Eukaryotic Pre-mRNAs As soon as RNA polymerase II has produced about 25 nucleotides of RNA, the 5ʹ end of the new RNA molecule is modified by addition of a cap that consists of a modified guanine nucleotide (see Figure 6–21B). Three enzymes, acting in succession, perform the capping reaction: one (a phosphatase) removes a phosphate from the 5ʹ end of the nascent RNA, another (a guanyl transferase) adds a GMP in


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Cell_Biology_Alberts. Figure 6–22 eukaryotic RNA polymerase II as an “RNA factory.” As the polymerase transcribes DNA into RNA, it carries RNA-processing proteins on its tail that are transferred to the nascent RNA at the appropriate time. The tail contains 52 tandem repeats of a seven-amino-acid sequence, and there are two serines in each repeat. The capping proteins first bind to the RNA polymerase tail when it is phosphorylated on Ser5 of the heptad repeat late in the process of transcription initiation (see Figure 6–15). This strategy ensures that the RNA molecule is efficiently capped as soon as its 5ʹ end emerges from the RNA polymerase. As the polymerase continues transcribing, its tail is extensively phosphorylated on the Ser2 positions by a kinase associated with the elongating polymerase and is eventually dephosphorylated at Ser5 positions. These further modifications attract splicing and 3ʹ-end processing proteins to the moving polymerase, positioning them to act on the newly synthesized RNA as


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Cell_Biology_Alberts. dephosphorylated at Ser5 positions. These further modifications attract splicing and 3ʹ-end processing proteins to the moving polymerase, positioning them to act on the newly synthesized RNA as it emerges from the RNA polymerase. There are many RNA-processing enzymes, and not all travel with the polymerase. For RNA splicing, for example, the tail carries only a few critical components; once transferred to an RNA molecule, they serve as a nucleation site for the remaining components.


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Cell_Biology_Alberts. When RNA polymerase II finishes transcribing a gene, it is released from DNA, soluble phosphatases remove the phosphates on its tail, and it can reinitiate transcription. Only the fully dephosphorylated form of RNA polymerase II is competent to begin RNA synthesis at a promoter. a reverse linkage (5ʹ to 5ʹ instead of 5ʹ to 3ʹ), and a third (a methyl transferase) adds a methyl group to the guanosine (Figure 6–23). Because all three enzymes bind to the RNA polymerase tail phosphorylated at the Ser5 position—the modification added by TFIIH during transcription initiation— they are poised to modify the 5ʹ end of the nascent transcript as soon as it emerges from the polymerase.


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Cell_Biology_Alberts. The 5ʹ-methyl cap signifies the 5ʹ end of eukaryotic mRNAs, and this landmark helps the cell to distinguish mRNAs from the other types of RNA molecules present in the cell. For example, RNA polymerases I and III produce uncapped RNAs during transcription, in part because these polymerases lack a CTD. In the nucleus, the cap binds a protein complex called CBC (cap-binding complex), which, as we discuss in subsequent sections, helps a future mRNA be further processed and exported. The 5ʹ-methyl cap also has an important role in the translation of mRNAs in the cytosol, as we discuss later in the chapter. 5˜ end of nascent RNA transcript


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Cell_Biology_Alberts. 5˜ end of nascent RNA transcript As discussed in Chapter 4, the protein-coding sequences of eukaryotic genes are typically interrupted by noncoding intervening sequences (introns). Discovered 5˜ 3˜ in 1977, this feature of eukaryotic genes came as a surprise to scientists, who had been, until that time, familiar only with bacterial genes, which typically consist of a continuous stretch of coding DNA that is directly transcribed into mRNA. In marked contrast, eukaryotic genes were found to be broken up into small pieces of coding sequence (expressed sequences or exons) interspersed with much longer intervening sequences or introns; thus, the coding portion of a eukaryotic gene is often only a small fraction of the length of the gene (Figure 6–24).


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Cell_Biology_Alberts. Both intron and exon sequences are transcribed into RNA. The intron sequences are removed from the newly synthesized RNA through the process of RNA splicing. The vast majority of RNA splicing that takes place in cells func- tions in the production of mRNA, and our discussion of splicing focuses on this so-called precursor-mRNA (or pre-mRNA) splicing. Only after 5ʹand 3ʹ-end pro- add methyl group to base cessing and splicing have taken place is such RNA termed mRNA.


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Cell_Biology_Alberts. Figure 6–23 The reactions that cap the 5ʹ end of each RNA molecule synthesized by RNA polymerase II. The final cap contains a novel 5ʹ-to-5ʹ add methyl group to ribose linkage between the positively charged 7-methyl G residue and the 5ʹ end of the RNA transcript (see Figure 6–21B). The letter N represents any one of the four ribonucleotides, although the nucleotide that starts an RNA chain is usually a purine (an A or a G). (After A.J. Shatkin, BioEssays 7:275–277, 1987. With permission from Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.) CH3 exons 2000 200,000 nucleotide pairs


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Cell_Biology_Alberts. Each splicing event removes one intron, proceeding through two sequential phosphoryl-transfer reactions known as transesterifications; these join two exons together while removing the intron between them as a “lariat” (Figure 6–25). The machinery that catalyzes pre-mRNA splicing is complex, consisting of five addi-tional RNA molecules and several hundred proteins, and it hydrolyzes many ATP molecules per splicing event. This complexity ensures that splicing is accurate, while at the same time being flexible enough to deal with the enormous variety of introns found in a typical eukaryotic cell. It may seem wasteful to remove large numbers of introns by RNA splicing. In attempting to explain why it occurs, scientists have pointed out that the exon– intron arrangement would seem to facilitate the emergence of new and useful proteins over evolutionary time scales. Thus, the presence of numerous introns in DNA allows genetic recombination to readily combine the exons of different genes,


Cell_Biology_Alberts

Cell_Biology_Alberts. the emergence of new and useful proteins over evolutionary time scales. Thus, the presence of numerous introns in DNA allows genetic recombination to readily combine the exons of different genes, enabling genes for new proteins to evolve more easily by the combination of parts of preexisting genes. The observation, described in Chapter 3, that many proteins in present-day cells resemble patchworks composed from a common set of protein domains, supports this idea (see pp. 121–122). RNA splicing also has a present-day advantage. The transcripts of many eukaryotic genes (estimated at 95% of genes in humans) are spliced in more than one way, thereby allowing the same gene to produce a corresponding set of dif-ferent proteins (Figure 6–26). Rather than being the wasteful process it may have seemed at first sight, RNA splicing enables eukaryotes to increase the coding potential of their genomes. We shall return to this idea again in this chapter and the next, but we first need to describe


Cell_Biology_Alberts

Cell_Biology_Alberts. at first sight, RNA splicing enables eukaryotes to increase the coding potential of their genomes. We shall return to this idea again in this chapter and the next, but we first need to describe the cellular machinery that performs this remarkable task. O(A) (B) 5˜5˜AOH AHO 3˜5˜exon sequence intron sequence 3˜exon 2˜


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Cell_Biology_Alberts. Figure 6–24 Structure of two human genes showing the arrangement of exons and introns. (A) The relatively small β-globin gene, which encodes a subunit of the oxygen-carrying protein hemoglobin, contains 3 exons (see also Figure 4–7). (B) The much larger Factor VIII gene contains 26 exons; it codes for a protein (Factor VIII) that functions in the blood-clotting pathway. The most prevalent form of hemophilia results from mutations in this gene.


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Cell_Biology_Alberts. Figure 6–25 The pre-mRNA splicing reaction. (A) In the first step, a specific adenine nucleotide in the intron sequence (indicated in red ) attacks the 5ʹ splice site and cuts the sugar-phosphate backbone of the RNA at this point. The cut 5ʹ end of the intron becomes covalently linked to the adenine nucleotide, as shown in detail in (B), thereby creating a loop in the RNA molecule. The released free 3ʹ-OH end of the exon sequence then reacts with the start of the next exon sequence, joining the two exons together and releasing the intron sequence in the shape of a lariat. The two exon sequences thereby become joined into a continuous coding sequence. The released intron sequence is eventually broken down into single nucleotides, which 5˜ 3˜ are recycled. TRANSCRIPTION, SPLICING, AND 3˜ CLEAVAGE/POLYADENYLATION


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Cell_Biology_Alberts. TRANSCRIPTION, SPLICING, AND 3˜ CLEAVAGE/POLYADENYLATION The mechanism of pre-mRNA splicing shown in Figure 6–24 requires that the splicing machinery recognize three portions of the precursor RNA molecule: the 5ʹ splice site, the 3ʹ splice site, and the branch point in the intron sequence that forms the base of the excised lariat. Not surprisingly, each site has a consensus nucleotide sequence that is similar from intron to intron and provides the cell with cues for where splicing is to take place (Figure 6–27). However, these consensus sequences are relatively short and can accommodate extensive sequence variability; as we shall see shortly, the cell incorporates additional types of information to ultimately choose exactly where, on each RNA molecule, splicing is to take place.


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Cell_Biology_Alberts. The high variability of the splicing consensus sequences presents a special challenge for scientists attempting to decipher genome sequences. Introns range in size from about 10 nucleotides to over 100,000 nucleotides, and choosing the precise borders of each intron is a difficult task even with the aid of powerful computers. The possibility of alternative splicing compounds the problem of predicting protein sequences solely from a genome sequence. This difficulty is one of the main barriers to identifying all of the genes in a complete genome sequence, and it is one of the primary reasons why we know only the approximate number of different proteins produced by the human genome. RNA Splicing Is Performed by the Spliceosome


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Cell_Biology_Alberts. RNA Splicing Is Performed by the Spliceosome Unlike the other steps of mRNA production we have discussed, key steps in RNA splicing are performed by RNA molecules rather than proteins. Specialized RNA molecules recognize the nucleotide sequences that specify where splicing is to occur and also catalyze the chemistry of splicing. These RNA molecules are relatively short (less than 200 nucleotides each), and there are five of them, U1, U2, U4, U5, and U6. Known as snRNAs (small nuclear RNAs), each is complexed with at least seven protein subunits to form an snRNP (small nuclear ribonucleoprotein). –°–°–°AG GURAGU –°– –°– °YURAC – ....°– YYYYYYYYNCAG G –°–°–° 5˜3˜ portion of a primary transcript exon 1 intron exon 2 portion of mRNA exon 1 exon 2 Figure 6–26 Alternative splicing of the α-tropomyosin gene from rat.


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Cell_Biology_Alberts. Figure 6–26 Alternative splicing of the α-tropomyosin gene from rat. α-Tropomyosin is a coiled-coil protein (see Figure 3–9) that carries out several tasks, most notably the regulation of contraction in muscle cells. The primary transcript can be spliced in different ways, as indicated in the figure, to produce distinct mRNAs, which then give rise to variant proteins. Some of the splicing patterns are specific for certain types of cells. For example, the α-tropomyosin made in striated muscle is different from that made from the same gene in smooth muscle. The arrowheads in the top part of the figure mark the sites where cleavage and poly-A addition form the 3ʹ ends of the mature mRNAs.


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Cell_Biology_Alberts. Figure 6–27 The consensus nucleotide sequences in an RNA molecule that signal the beginning and the end of most introns in humans. The three blocks of nucleotide sequences shown are required to remove an intron sequence. Here A, G, U, and C are the standard RNA nucleotides; R stands for purines (A or G); and Y stands for pyrimidines (C or U). The A highlighted in red forms the branch point of the lariat produced by splicing (see Figure 6–25). Only the GU at the start of the intron and the AG at its end are invariant nucleotides in the splicing consensus sequences. Several different nucleotides can occupy the remaining positions, although the indicated nucleotides are preferred. The distances along the RNA between the three splicing consensus sequences are highly variable; however, the distance between the branch point and 3ʹ splice junction is typically much shorter than that between the 5ʹ splice junction and the branch point.


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Cell_Biology_Alberts. These snRNPs form the core of the spliceosome, the large assembly of RNA and protein molecules that performs pre-mRNA splicing in the cell. During the splicing reaction, recognition of the 5ʹ splice junction, the branch-point site, and the 3ʹsplice junction is performed largely through base-pairing between the snRNAs and the consensus RNA sequences in the pre-mRNA substrate. The spliceosome is a complex and dynamic machine. When studied in vitro, a few components of the spliceosome assemble on pre-mRNA and, as the splicing reaction proceeds, new components enter and those that have already performed their tasks are jettisoned (Figure 6–28). However, many scientists believe that, inside the cell, the spliceosome is a preexisting, loose assembly of all the components—capturing, splicing, and releasing RNA as a coordinated unit, and undergoing extensive rearrangements each time a splice is made. intron portion of a 5˜ 3˜ pre-mRNA transcript


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Cell_Biology_Alberts. intron portion of a 5˜ 3˜ pre-mRNA transcript U1, U4 excised intron sequence in the form of a lariat 3˜ (intron RNA will be degraded in the nucleus; snRNPs will exon 1 exon 2 The U1 snRNP forms base pairs with the 5˜ splice junction (see Figure 6–29) and the BBP (branch-point binding protein) and U2AF (U2 auxilliary factor) recognize the branch-point site. The U2 snRNP displaces BBP and U2AF and forms base pairs with the branch-point site consensus sequence. The U4/U6•U5 “triple” snRNP enters the reaction. In this triple snRNP, the U4 and U6 snRNAs are held frmly together by base-pair interactions. Subsequent rearrangements break apart the U4/U6 base pairs, allowing U6 to displace U1 at the 5˜ splice junction (see Figure 6–29). This creates the active site that catalyzes the frst phosphoryl-transferase reaction. Additional RNA–RNA rearrangements create the active site for the second phosphoryl-transferase reaction, which then completes the splice (see Figure 6–25A).


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Cell_Biology_Alberts. Additional RNA–RNA rearrangements create the active site for the second phosphoryl-transferase reaction, which then completes the splice (see Figure 6–25A). Figure 6–28 The pre-mRNA splicing mechanism. RNA splicing is catalyzed by an assembly of snRNPs (shown as colored circles) plus other proteins (most of which are not shown), which together constitute the spliceosome. The spliceosome recognizes the splicing signals on a pre-mRNA molecule, brings the two ends of the intron together, and provides the enzymatic activity for the two reaction steps required (see Figure 6–25A and Movie 6.5). As indicated, a set of proteins called the exon junction complex (EJC) remains on the spliced mRNA molecule; its subsequent role will be discussed shortly. The Spliceosome Uses ATP Hydrolysis to Produce a Complex Series of RNA–RNA Rearrangements


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Cell_Biology_Alberts. The Spliceosome Uses ATP Hydrolysis to Produce a Complex Series of RNA–RNA Rearrangements ATP hydrolysis is not required for the chemistry of RNA splicing per se since the two transesterification reactions preserve the high-energy phosphate bonds. However, extensive ATP hydrolysis is required for the assembly and rearrangements of the spliceosome. Some of the additional proteins that make up the spliceosome use the energy of ATP hydrolysis to break existing RNA–RNA interactions to allow the formation of new ones. Each successful splice requires approximately 200 proteins, if we include those that form the snRNPs.


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Cell_Biology_Alberts. What is the purpose of these rearrangements? First, they allow the splicing signals on the pre-RNA to be examined by snRNPs several times during the course of splicing. For example, the U1 snRNP initially recognizes the 5ʹ splice site through conventional base-pairing; as splicing proceeds, these base pairs are broken (using the energy of ATP hydrolysis) and U1 is replaced by U6 (Figure 6–29). This type of RNA–RNA rearrangement (in which the formation of one RNA–RNA interaction requires the disruption of another) occurs several times during splicing and allows the spliceosomes to check and recheck the splicing signals, thereby increasing the overall accuracy of splicing. Second, the rearrangements that take place in the spliceosome create the active sites for the two transesterification reactions. These two active sites are created, one after the other, and only after the splicing signals on the pre-mRNA have been checked several times. This orderly progression ensures that splicing


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Cell_Biology_Alberts. reactions. These two active sites are created, one after the other, and only after the splicing signals on the pre-mRNA have been checked several times. This orderly progression ensures that splicing accidents occur only rarely.


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Cell_Biology_Alberts. One of the most surprising features of the spliceosome is the nature of the catalytic sites: they are formed by both protein and RNA molecules, although the RNA molecules catalyze the actual chemistry of splicing. In the last section of this chapter, we discuss in general terms the structural and chemical properties of RNA molecules that allow them to act as catalysts.


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Cell_Biology_Alberts. Once the splicing chemistry is completed, the snRNPs remain bound to the lariat. The disassembly of these snRNPs from the lariat (and from each other) requires another series of RNA–RNA rearrangements that require ATP hydrolysis, thereby returning the snRNAs to their original configuration so that they can be used again in a new reaction. At the completion of a splice, the spliceosome directs a set of proteins to bind to the mRNA near the position formerly occupied by the intron. Called the exon junction complex (EJC), these proteins mark the site of a successful splicing event and, as we shall see later in this chapter, influence the subsequent fate of the mRNA. Other Properties of Pre-mRNA and Its Synthesis Help to Explain the Choice of Proper Splice Sites


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Cell_Biology_Alberts. Other Properties of Pre-mRNA and Its Synthesis Help to Explain the Choice of Proper Splice Sites As we have seen, intron sequences vary enormously in size, with some being in excess of 100,000 nucleotides. If splice-site selection were determined solely by the snRNPs acting on a preformed, protein-free RNA molecule, we would expect frequent splicing mistakes—such as exon skipping and the use of “cryptic” splice sites (Figure 6–30). The fidelity mechanisms built into the spliceosome to suppress errors, however, are supplemented by two additional strategies that further increase the accuracy of splicing. The first is a simple consequence of splicing being coupled to transcription. As transcription proceeds, the phosphorylated tail of RNA polymerase carries several components of the spliceosome (see Figure Figure 6–29 One of the many rearrangements that take place in the spliceosome during pre-mRNA splicing.


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Cell_Biology_Alberts. Figure 6–29 One of the many rearrangements that take place in the spliceosome during pre-mRNA splicing. This example comes from the yeast Saccharomyces cerevisiae, in which the nucleotide sequences involved are slightly different from those in human cells. The exchange of U1 snRNP for U6 snRNP occurs just before the first phosphoryltransfer reaction (see Figure 6–28). This exchange requires the 5ʹ splice site to be read by two different snRNPs, thereby increasing the accuracy of 5ʹ splice-site selection by the spliceosome. portion of exon 2 exon 1 exon 3 exon 1 6–22), and these components are transferred directly from the polymerase to the


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Cell_Biology_Alberts. portion of exon 2 exon 1 exon 3 exon 1 6–22), and these components are transferred directly from the polymerase to the RNA as the RNA emerges from the polymerase. This strategy helps the cell keep track of introns and exons: for example, the snRNPs that assemble at a 5ʹ splice site are initially presented only with the single 3ʹ splice site that emerges next from the polymerase; the potential sites further downstream have not yet been synthe sized. The coordination of transcription with splicing is especially important in preventing inappropriate exon skipping.


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Cell_Biology_Alberts. A strategy called “exon definition” also helps cells choose the appropriate splice sites. Exon size tends to be much more uniform than intron size, averaging about 150 nucleotide pairs across a wide variety of eukaryotic organisms (Figure 6–31). Through exon definition, the splicing machinery can seek out the relatively homogeneously sized exon sequences. As RNA synthesis proceeds, a group of additional components (most notably SR proteins, so-named because they con help to mark off each 3ʹand 5ʹ splice site, starting at the 5ʹ end of the RNA (Figure 6–32). These proteins, in turn, recruit U1 snRNA, which marks the downstream exon boundary, and U2 snRNA, which specifies the upstream one. By specifically marking the exons in this way and thereby taking advantage of the relatively uni form size of exons, the cell increases the accuracy with which it deposits the initial splicing components on the nascent RNA and thereby avoids “near miss” splice sites. How the SR proteins discriminate


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Cell_Biology_Alberts. size of exons, the cell increases the accuracy with which it deposits the initial splicing components on the nascent RNA and thereby avoids “near miss” splice sites. How the SR proteins discriminate exon sequences from intron sequences is not understood in detail; however, it is known that some of the SR proteins bind preferentially to specific RNA sequences in exons, termed splicing enhancers.


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Cell_Biology_Alberts. In principle, since any one of several different codons can be used to code for a given amino acid, there is freedom to evolve the exon nucleotide sequence so as to form a binding site for an SR protein, without necessarily affecting the amino acid sequence that the exon specifies. Both the marking of exon and intron boundaries and the assembly of the spli ceosome begin on an RNA molecule while it is still being elongated by RNA poly merase at its 3ʹ end. However, the actual chemistry of splicing can take place later. This delay means that intron sequences are not necessarily removed from a pre mRNA molecule in the order in which they occur along the RNA chain. Figure 6–30 Two types of splicing errors. (A) Exon skipping. (B) Cryptic splice-site selection. Cryptic splicing signals are nucleotide sequences of RNA that closely resemble true splicing signals and are sometimes mistakenly used by the spliceosome.


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Cell_Biology_Alberts. Figure 6–31 Variation in intron and exon lengths in the human, worm, and fly genomes. (A) Size distribution of exons. (B) Size distribution of introns. Note that exon length is much more uniform than intron length. (Adapted from International Human Genome Sequencing Consortium, Nature 409:860–921, 2001. With permission from Macmillan Publishers Ltd.) percentage of introns percentage of exons 100 200 300 400 500 600 700 800 900 1000 <100 100–2000 2000–5000 5000–30,000 >30,000 exon length (nucleotide pairs) intron length (nucleotide pairs)


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Cell_Biology_Alberts. Although it may seem at first counterintuitive, the way a gene is packaged into chromatin can affect how the RNA transcript of that gene is ultimately spliced. Nucleosomes tend to be positioned over exons (which are, on average, close to the length of DNA in a nucleosome), and it has been proposed that these act as “speed bumps,” allowing the proteins responsible for exon definition to assemble on the RNA as it emerges from the polymerase. In addition, changes in chromatin structure are used to alter splicing patterns. There are two ways this can happen. First, because splicing and transcription are coupled, the rate at which RNA polymerase moves along DNA can affect RNA splicing. For example, if polymerase is moving slowly, exon skipping (see Figure 6–30A) is minimized: assembly of the initial spliceosome may be complete before an alternative choice of splice site even emerges from the RNA polymerase. The nucleosomes in condensed chromatin can cause polymerase to pause; the pattern


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Cell_Biology_Alberts. initial spliceosome may be complete before an alternative choice of splice site even emerges from the RNA polymerase. The nucleosomes in condensed chromatin can cause polymerase to pause; the pattern of pauses in turn affects the extent of RNA exposed at any given time to the splicing machinery.


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Cell_Biology_Alberts. There is a second and more direct way that chromatin structure can affect RNA splicing. Although the details are not yet understood, specific histone modifications attract components of the spliceosome, and, because the chromatin being transcribed is in close association with the nascent RNA, these splicing components can easily be transferred to the emerging RNA. In this way, certain types of histone modifications can affect the final pattern of splicing.


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Cell_Biology_Alberts. We have seen that the choice of splice sites depends on such features of the premRNA transcript as the strength of the three signals on the RNA (the 5ʹand 3ʹ splice junctions and the branch point) for the splicing machinery, the co-transcriptional assembly of the spliceosome, chromatin structure, and the “bookkeeping” that underlies exon definition. We do not know exactly how accurate splicing normally is because, as we see later, there are several quality control systems that rapidly destroy mRNAs whose splicing goes awry. However, we do know that, compared with other steps in gene expression, splicing is unusually flexible.


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Cell_Biology_Alberts. Thus, for example, a mutation in a nucleotide sequence critical for splicing of a particular intron does not necessarily prevent splicing of that intron altogether. Instead, the mutation typically creates a new pattern of splicing (Figure 6–33). Most commonly, an exon is simply skipped (Figure 6–33B). In other cases, the mutation causes a cryptic splice junction to be efficiently used (Figure 6–33C). Apparently, the splicing machinery has evolved to pick out the best possible pattern of splice junctions, and if the optimal one is damaged by mutation, it will seek out the next best pattern, and so on. This inherent plasticity in the process of RNA splicing suggests that changes in splicing patterns caused by random mutations have been important in the evolution of genes and organisms. It also means that mutations that affect splicing can be severely detrimental to the organism: in addition to the β thalassemia, example presented in Figure 6–33, aberrant


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Cell_Biology_Alberts. Figure 6–32 The exon definition hypothesis. According to this idea, SR proteins bind to each exon sequence in the pre-mRNA and thereby help to guide the snRNPs to the proper intron/ exon boundaries. This demarcation of exons by the SR proteins occurs cotranscriptionally, beginning at the CBC (cap-binding complex) at the 5ʹ end. It has been proposed that a group of proteins known as the heterogeneous nuclear ribonucleoproteins (hnRNPs) may preferentially associate with intron sequences, further helping the spliceosome distinguish introns from exons. (Adapted from R. Reed, Curr. Opin. Cell Biol. 12:340–345, 2000. With permission from Elsevier.) splicing plays important roles in the development of cystic fibrosis, frontotemporal dementia, Parkinson’s disease, retinitis pigmentosa, spinal muscular atrophy, myotonic dystrophy, premature aging, and cancer. It has been estimated that of the many point mutations that cause inherited human diseases, 10% produce aberrant splicing of the gene


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Cell_Biology_Alberts. atrophy, myotonic dystrophy, premature aging, and cancer. It has been estimated that of the many point mutations that cause inherited human diseases, 10% produce aberrant splicing of the gene containing the mutation.


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Cell_Biology_Alberts. The plasticity of RNA splicing also means that the cell can easily regulate the pattern of RNA splicing. Earlier in this section we saw that alternative splicing can give rise to different proteins from the same gene and that this is a common strategy to enhance the coding potential of genomes. Some examples of alternative splicing are constitutive; that is, the alternatively spliced mRNAs are produced continuously by cells of an organism. However, in many cases, the cell regulates the splicing patterns so that different forms of the protein are produced at different times and in different tissues (see Figure 6–26). In Chapter 7, we return to this issue to discuss some specific examples of regulated RNA splicing.


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Cell_Biology_Alberts. When the spliceosome was first discovered, it puzzled molecular biologists. Why do RNA molecules instead of proteins perform important roles in splice-site recognition and in the chemistry of splicing? Why is a lariat intermediate used rather than the apparently simpler alternative of bringing the 5ʹand 3ʹ splice sites together in a single step, followed by their direct cleavage and rejoining? The answers to these questions reflect the way in which the spliceosome has evolved.


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Cell_Biology_Alberts. As discussed briefly in Chapter 1 (and in more detail in the final section of this chapter), it is likely that early cells used RNA molecules rather than proteins as their major catalysts and that they stored their genetic information in RNA rather than in DNA sequences. RNA-catalyzed splicing reactions presumably had critical roles in these early cells. As evidence, some self-splicing RNA introns (that is, intron sequences in RNA whose splicing out can occur in the absence of proteins or any other RNA molecules) remain today—for example, in the nuclear rRNA genes of the ciliate Tetrahymena, in a few bacteriophage T4 genes, and in some mitochondrial and chloroplast genes. In these cases, the RNA molecule folds into a specific three-dimensional structure that brings the intron/exon junctions together and catalyzes the two transesterification reactions. A self-splicing intron sequence can be identified in a test tube by incubating a pure RNA molecule that contains the intron sequence


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Cell_Biology_Alberts. together and catalyzes the two transesterification reactions. A self-splicing intron sequence can be identified in a test tube by incubating a pure RNA molecule that contains the intron sequence and observing the splicing reaction. Because the basic chemistry of some self-splicing reactions is so similar to pre-mRNA splicing, it has been proposed that the much more involved process of pre-mRNA splicing evolved from a simpler, ancestral form of RNA self-splicing.


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Cell_Biology_Alberts. RNA-Processing Enzymes Generate the 3ʹ End of Eukaryotic mRNAs We have seen that the 5ʹ end of the pre-mRNA produced by RNA polymerase II is capped almost as soon as it emerges from the RNA polymerase. Then, as the polymerase continues its movement along a gene, the spliceosome assembles on the RNA and delineates the intron and exon boundaries. The long C-terminal tail of the RNA polymerase coordinates these processes by transferring capping and splicing components directly to the RNA as it emerges from the enzyme. In this section, we shall see that, as RNA polymerase II reaches the end of a gene, a similar mechanism ensures that the 3ʹ end of the pre-mRNA is appropriately processed.


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Cell_Biology_Alberts. The position of the 3ʹ end of each mRNA molecule is specified by signals encoded in the genome (Figure 6–34). These signals are transcribed into RNA as the RNA polymerase II moves through them, and they are then recognized (as RNA) by a series of RNA-binding proteins and RNA-processing enzymes (Figure 6–35). Two multisubunit proteins, called CstF (cleavage stimulation factor) and CPSF (cleavage and polyadenylation specificity factor), are of special importance. intron sequences normal mRNA is formed from three exons A SINGLE-NUCLEOTIDE CHANGE THAT DESTROYS A NORMAL SPLICE SITE, THEREBY CAUSING EXON SKIPPING mRNA with exon 2 missing A SINGLE-NUCLEOTIDE CHANGE THAT DESTROYS A NORMAL SPLICE SITE, THEREBY ACTIVATING A CRYPTIC SPLICE SITE mRNA with extended exon 3 mRNA with extra exon inserted between exon 2 and exon 3 Figure 6–33 Abnormal processing of the β-globin primary RNA transcript in humans with the disease β thalassemia.


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Cell_Biology_Alberts. Figure 6–33 Abnormal processing of the β-globin primary RNA transcript in humans with the disease β thalassemia. In the examples shown, the disease (a severe anemia due to aberrant hemoglobin synthesis) is caused by splice-site mutations found in the genomes of affected patients. The dark blue boxes represent the three normal exon sequences; the red lines connect the 5ʹ and 3ʹ splice sites that are used. In (B), (C), and (D), the light blue boxes depict new nucleotide sequences included in the final mRNA molecule as a result of the mutation denoted by the black arrowhead. Note that when a mutation leaves a normal splice site without a partner, an exon is skipped (B) or one or more abnormal cryptic splice sites nearby is used as the partner site (C). [Adapted in part from S.H. Orkin, in The Molecular Basis of Blood Diseases (G. Stamatoyannopoulos et al., eds.), pp. 106–126. Philadelphia: Saunders, 1987.] degraded in the nucleus


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Cell_Biology_Alberts. Both of these proteins travel with the RNA polymerase tail and are transferred to the 3ʹ-end processing sequence on an RNA molecule as it emerges from the RNA polymerase.


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Cell_Biology_Alberts. Once CstF and CPSF bind to their recognition sequences on the emerging RNA molecule, additional proteins assemble with them to create the 3ʹend of the mRNA. First, the RNA is cleaved from the polymerase (see Figure 6–35). Next an enzyme called poly-A polymerase (PAP) adds, one at a time, approximately 200 A nucleotides to the 3ʹ end produced by the cleavage. The nucleotide precursor for these additions is ATP, and the same type of 5ʹ-to-3ʹ bonds are formed as in conventional RNA synthesis. But unlike other RNA polymerases, poly-A polymerase does not require a template; hence the poly-A tail of eukaryotic mRNAs is not directly encoded in the genome. As the poly-A tail is synthesized, proteins called poly-A-binding proteins assemble onto it and, by a poorly understood mechanism, help determine the final length of the tail.


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Cell_Biology_Alberts. After the 3ʹ-end of a eukaryotic pre-mRNA molecule has been cleaved, the RNA polymerase II continues to transcribe, in some cases for hundreds of nucleotides. Once 3ʹ-end cleavage has occurred, the newly synthesized RNA that emerges from the polymerases lacks a 5ʹ cap; this unprotected RNA is rapidly degraded by a 5ʹ →3ʹ exonuclease carried along on the polymerase tail. Apparently, it is this continued RNA degradation that eventually causes the RNA polymerase to release its grip on the template and terminate transcription. Mature Eukaryotic mRNAs Are Selectively Exported from the Nucleus


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Cell_Biology_Alberts. Mature Eukaryotic mRNAs Are Selectively Exported from the Nucleus Eukaryotic pre-mRNA synthesis and processing take place in an orderly fashion within the cell nucleus. But of the pre-mRNA that is synthesized, only a small fraction—the mature mRNA—is of further use to the cell. Most of the rest—excised introns, broken RNAs, and aberrantly processed pre-mRNAs—is not only useless but potentially dangerous. How does the cell distinguish between the relatively rare mature mRNA molecules it wishes to keep and the overwhelming amount of debris created by RNA processing?