Cell_Biology_Alberts

Cell_Biology_Alberts. The answer is that, as an RNA molecule is processed, it loses certain proteins and acquires others. For example, we have seen that acquisition of cap-binding complexes, exon junction complexes, and poly-A-binding proteins mark the completion of capping, splicing, and poly-A addition, respectively. A properly completed mRNA molecule is also distinguished by the proteins it lacks. For example, the presence of an snRNP protein would signify incomplete or aberrant splicing. Only when the proteins present on an mRNA molecule collectively signify that processing was successfully completed is the mRNA exported from the nucleus into the cytosol, where it can be translated into protein. Improperly processed mRNAs


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–35 Some of the major steps in generating the 3ʹ end of a eukaryotic mRNA. This process is much more complicated than the analogous process in bacteria, where the RNA polymerase simply stops at a termination signal and releases both the 3ʹ end of its transcript and the DNA template (see Figure 6–11). Figure 6–34 Consensus nucleotide sequences that direct cleavage and polyadenylation to form the 3ʹ end of a eukaryotic mRNA. These sequences are encoded in the genome, and specific proteins recognize them—as RNA—after they are transcribed. As shown in Figure 6–35, the hexamer AAUAAA is bound by CPSF and the GU-rich element beyond the cleavage site is bound by CstF; the CA sequence is bound by a third protein factor required for the cleavage step. Like other consensus nucleotide sequences discussed in this chapter (see Figure 6–12), the sequences shown in the figure represent a variety of individual cleavage and polyadenylation signals.


Cell_Biology_Alberts

Cell_Biology_Alberts. mature 3˜ end of an mRNA molecule and other RNA debris (excised intron sequences, for example) are retained in the nucleus, where they are eventually degraded by the nuclear exosome, a large protein complex whose interior is rich in 3ʹ-to-5ʹ RNA exonucleases (Figure 6–36). Eukaryotic cells thus export only useful RNA molecules to the cytoplasm, while debris is disposed of in the nucleus.


Cell_Biology_Alberts

Cell_Biology_Alberts. Of all the proteins that assemble on pre-mRNA molecules as they emerge from transcribing RNA polymerases, the most abundant are the hnRNPs (heterogeneous nuclear ribonuclear proteins). Some of these proteins (there are approximately 30 different ones in humans) unwind the hairpin helices in the RNA so that splicing and other signals on the RNA can be read more easily. Others preferentially package the RNA contained in the very long intron sequences typical in complex organisms (see Figure 6–31) and these may play an important role in distinguishing mature mRNA from the debris left over from RNA processing.


Cell_Biology_Alberts

Cell_Biology_Alberts. Successfully processed mRNAs are guided through the nuclear pore complexes (NPCs)—aqueous channels in the nuclear membrane that directly connect the nucleoplasm and cytosol (Figure 6–37). Small molecules (less than 60,000 daltons) can diffuse freely through these channels. However, most of the macromolecules in cells, including mRNAs complexed with proteins, are far too large to pass through the channels without a special process. The cell uses energy to actively transport such macromolecules in both directions through the nuclear pore complexes.


Cell_Biology_Alberts

Cell_Biology_Alberts. As explained in detail in Chapter 12, macromolecules are moved through nuclear pore complexes by nuclear transport receptors, which, depending on the identity of the macromolecule, escort it from the nucleus to the cytoplasm or vice versa. For mRNA export to occur, a specific nuclear transport receptor must be loaded onto the mRNA, a step that, in many organisms, takes place in concert with 3ʹ cleavage and polyadenylation. Once it helps to move an RNA molecule through the nuclear pore complex, the transport receptor dissociates from the mRNA, re-enters the nucleus, and is then used to export a new mRNA molecule.


Cell_Biology_Alberts

Cell_Biology_Alberts. The export of mRNA–protein complexes from the nucleus can be readily observed with the electron microscope for the unusually abundant mRNA of the insect Balbiani Ring genes. As these genes are transcribed, the newly formed RNA is seen to be packaged by proteins, including hnRNPs, SR proteins, and components of the spliceosome. This protein–RNA complex undergoes a series of structural transitions, probably reflecting RNA processing events, culminating in a curved fiber (see Figure 6–37). This curved fiber moves through the nucleoplasm and enters the nuclear pore complex (with its 5ʹ cap proceeding first), and it then undergoes another series of structural transitions as it moves through the pore. These and other observations reveal that the pre-mRNA–protein and mRNA–protein complexes are dynamic structures that gain and lose numerous specific proteins during RNA synthesis, processing, and export (Figure 6–38).


Cell_Biology_Alberts

Cell_Biology_Alberts. The analysis just described has been complemented by new methods that allow researchers to track the fate of more typical mRNA molecules, which can RNA as it emerges from RNA polymerase


Cell_Biology_Alberts

Cell_Biology_Alberts. RNA as it emerges from RNA polymerase Figure 6–36 Structure of the core of human RNA exosome. RNA is fed into one end of the central pore and is degraded by RNAses that associate with the other end. Nine different protein subunits (each represented by a different color) make up this large ring structure. Eukaryotic cells have both a nuclear exosome and a cytoplasmic exosome; both forms include the core exosome shown here and additional subunits (including specialized RNAses) that differentiate the two forms. The nuclear exosome degrades aberrant RNAs before they are exported to the cytosol. It also processes certain types of RNA (for example, the ribosomal RNAs) to produce their final form. The cytoplasmic form of the exosome is responsible for degrading mRNAs in the cytosol, and is thus crucial in determining the lifetime of each mRNA molecule. (PDB code: 2NN6.)


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–37 Transport of a large mRNA molecule through the nuclear pore complex. (A) The maturation of an mRNA molecule as it is synthesized by RNA polymerase and packaged by a variety of nuclear proteins. This drawing of an unusually large and abundant insect RNA, called the Balbiani Ring mRNA, is based on electron microscope micrographs such as that shown in (B). (A, adapted from B. Daneholt, Cell 88:585–588, 1997. With permission from Elsevier; B, from B.J. Stevens and H. Swift, J. Cell Biol. 31:55–77, 1966. With permission from The Rockefeller University Press.) be fluorescently labeled and observed individually. A typical RNA molecule is released from its site of transcription and spends several minutes diffusing to a nuclear pore complex. During this time it is likely that RNA processing events continue and that the RNA sheds previously bound proteins and acquires new ones. Once it arrives at the entrance to the pore, the “export-ready” mRNA hovers for several seconds, during


Cell_Biology_Alberts

Cell_Biology_Alberts. events continue and that the RNA sheds previously bound proteins and acquires new ones. Once it arrives at the entrance to the pore, the “export-ready” mRNA hovers for several seconds, during which time the completion of processing may occur, and then is transported through the pore very rapidly, in tens of milliseconds. Some mRNA–protein complexes are very large, and how they move through the nuclear pore complexes so rapidly remains a mystery.


Cell_Biology_Alberts

Cell_Biology_Alberts. Some of the proteins deposited on the mRNA while it is still in the nucleus can affect the fate of the RNA after it is transported to the cytosol. Thus, the stability of an mRNA in the cytosol, the efficiency with which it is translated into protein, and its ultimate destination in the cytosol can all be determined by proteins acquired in the nucleus that remain bound to the RNA after it leaves the nucleus. But before discussing what happens to mRNAs in the cytosol, we briefly consider how the synthesis and processing of some noncoding RNA molecules occurs. There are many types of noncoding RNAs produced by cells (see Table 6–1, p. 305), but here we focus on the rRNAs, which are critically important for the translation of mRNAs into protein. Noncoding RNAs Are Also Synthesized and Processed in the Nucleus


Cell_Biology_Alberts

Cell_Biology_Alberts. Noncoding RNAs Are Also Synthesized and Processed in the Nucleus Only a few percent of the dry weight of a mammalian cell is RNA; of that, only about 3–5% is mRNA. The bulk of the RNA in cells performs structural and catalytic functions (see Table 6–1). The most abundant RNAs in cells are the ribosomal RNAs (rRNAs), constituting approximately 80% of the RNA in rapidly dividing cells. As discussed later in this chapter, these RNAs form the core of the ribosome. Unlike bacteria—in which a single RNA polymerase synthesizes all RNAs in the cell—eukaryotes have a separate, specialized polymerase, RNA polymerase I, that is dedicated to producing rRNAs. RNA polymerase I is similar structurally to the RNA polymerase II discussed previously; however, the absence of a C-terminal tail in polymerase I helps to explain why its transcripts are neither capped nor polyadenylated.


Cell_Biology_Alberts

Cell_Biology_Alberts. Because multiple rounds of translation of each mRNA molecule can provide an enormous amplification in the production of protein molecules, many of the proteins that are very abundant in a cell can be synthesized from genes that are present in a single copy per haploid genome (see Figure 6–3). In contrast, the RNA components of the ribosome are final gene products, and a growing mammalian cell must synthesize approximately 10 million copies of each type of ribosomal RNA in each cell generation to construct its 10 million ribosomes. The cell can produce adequate quantities of ribosomal RNAs only because it contains multiple copies of the rRNA genes that code for ribosomal RNAs (rRNAs). Even E. coli needs seven copies of its rRNA genes to meet the cell’s need for ribosomes. Human cells contain about 200 rRNA gene copies per haploid genome, spread Figure 6–38 Schematic illustration of an export-ready mRNA molecule and its transport through the nuclear pore.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–38 Schematic illustration of an export-ready mRNA molecule and its transport through the nuclear pore. As indicated, some proteins travel with the mRNA as it moves through the pore, whereas others remain in the nucleus. The nuclear export receptor for mRNAs is a complex of proteins that binds to an mRNA molecule once it has been correctly spliced and polyadenylated. After the mRNA has been exported to the cytosol, this export receptor dissociates from the mRNA and is re-imported into the nucleus, where it can be used again. The final check indicated here, called nonsense-mediated decay, will be described later in the chapter. out in small clusters on five different chromosomes (see Figure 4–11), while cells of the frog Xenopus contain about 600 rRNA gene copies per haploid genome in a single cluster on one chromosome (Figure 6–39).


Cell_Biology_Alberts

Cell_Biology_Alberts. There are four types of eukaryotic rRNAs, each present in one copy per ribosome. Three of the four rRNAs (18S, 5.8S, and 28S) are made by chemically modifying and cleaving a single large precursor rRNA (Figure 6–40); the fourth (5S RNA) is synthesized from a separate cluster of genes by a different polymerase, RNA polymerase III, and does not require chemical modification.


Cell_Biology_Alberts

Cell_Biology_Alberts. Extensive chemical modifications occur in the 13,000-nucleotide-long precursor rRNA before the rRNAs are cleaved out of it and assembled into ribosomes. These include about 100 methylations of the 2ʹ-OH positions on nucleotide sugars and 100 isomerizations of uridine nucleotides to pseudouridine (Figure 6–41A). The functions of these modifications are not understood in detail, but they probably aid in the folding and assembly of the final rRNAs, or subtly alter the function of ribosomes. Each modification is made at a specific position in the precursor rRNA, specified by “guide RNAs,” which position themselves on the precursor rRNA through base-pairing and thereby bring an RNA-modifying enzyme to the appropriate position (Figure 6–41B). Other guide RNAs promote cleavage of the precursor rRNAs into the mature rRNAs, probably by causing conformational changes in the precursor rRNA that expose these sites to nucleases. All of these guide RNAs are members of a large class of RNAs called


Cell_Biology_Alberts

Cell_Biology_Alberts. rRNAs into the mature rRNAs, probably by causing conformational changes in the precursor rRNA that expose these sites to nucleases. All of these guide RNAs are members of a large class of RNAs called small nucleolar RNAs (or snoRNAs), so named because these RNAs perform their functions in a sub-compartment of the nucleus called the nucleolus. Many snoRNAs are encoded in 13,000 nucleotides degraded regions of nucleotide sequence


Cell_Biology_Alberts

Cell_Biology_Alberts. CLEAVAGE 5.8S rRNA 28S rRNA 5S rRNA made elsewhere incorporated into large ribosomal subunit Figure 6–39 Transcription from tandemly arranged rRNA genes, as seen in the electron microscope. The pattern of alternating transcribed gene and nontranscribed spacer is readily seen. A higher-magnification view of rRNA genes is shown in Figure 6–10. (From V.E. Foe, Cold Spring Harb. Symp. Quant. Biol. 42:723–740, 1978. With permission from Cold Spring Harbor Laboratory Press.) Figure 6–40 The chemical modification and nucleolytic processing of a eukaryotic 45S precursor rRNA molecule into three separate ribosomal RNAs.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–40 The chemical modification and nucleolytic processing of a eukaryotic 45S precursor rRNA molecule into three separate ribosomal RNAs. Two types of chemical modifications (color-coded as indicated in Figure 6–41) are made to the precursor rRNA before it is cleaved. Nearly half of the nucleotide sequences in this precursor rRNA are discarded and degraded in the nucleus by the exosome. The rRNAs are named according to their “S” values, which refer to their rate of sedimentation in an ultracentrifuge. The larger the S value, the larger the rRNA. the introns of other genes, especially those encoding ribosomal proteins. They are synthesized by RNA polymerase II and processed from excised intron sequences. The Nucleolus Is a Ribosome-Producing Factory


Cell_Biology_Alberts

Cell_Biology_Alberts. The Nucleolus Is a Ribosome-Producing Factory The nucleolus is the most obvious structure seen in the nucleus of a eukaryotic cell when viewed in the light microscope. It was so closely scrutinized by early cytologists that an 1898 review could list some 700 references. We now know that the nucleolus is the site for the processing of rRNAs and their assembly into ribosome subunits. Unlike many of the major organelles in the cell, the nucleolus is not bound by a membrane (Figure 6–42); instead, it is a huge aggregate Figure 6–41 Modifications of the precursor rRNA by guide RNAs. (A) Two prominent covalent modifications made to rRNA; the differences from the initially incorporated nucleotide are indicated by red atoms. Pseudouridine is an isomer of uridine; the base has been “rotated,” and is attached to the red C rather than to the red N of the sugar (compare to Figure 6–5B).


Cell_Biology_Alberts

Cell_Biology_Alberts. (B) As indicated, snoRNAs determine the sites of modification by base-pairing to complementary sequences on the precursor rRNA. The snoRNAs are bound to proteins, and the complexes are called snoRNPs (small nucleolar ribonucleoproteins). snoRNPs contain both the guide sequences and the enzymes that modify the rRNA. Figure 6–42 electron micrograph of a thin section of a nucleolus in a human fibroblast, showing its three distinct zones. (A) View of entire nucleus. Higher-power view of the nucleolus. It is believed that transcription of the rRNA genes takes place between the fibrillar center and the dense fibrillar component and that processing of the rRNAs and their assembly into the two subunits of the ribosome proceeds outward from the dense fibrillar component to the surrounding granular components. (Courtesy of E.G. Jordan and J. McGovern.)


Cell_Biology_Alberts

Cell_Biology_Alberts. J. McGovern.) Figure 6–43 Changes in the appearance of the nucleolus in a human cell during the cell cycle. Only the cell nucleus is represented in this diagram. In most eukaryotic cells, the nuclear envelope breaks down during mitosis, as indicated by the dashed circles. of macromolecules, including the rRNA genes themselves, precursor rRNAs, mature rRNAs, rRNA-processing enzymes, snoRNPs, a large set of assembly factors (including ATPases, GTPases, protein kinases, and RNA helicases), ribosomal proteins, and partly assembled ribosomes. The close association of all these components allows the assembly of ribosomes to occur rapidly and smoothly.


Cell_Biology_Alberts

Cell_Biology_Alberts. Various types of RNA molecules play a central part in the chemistry and structure of the nucleolus, suggesting that it may have evolved from an ancient structure present in cells dominated by RNA catalysis. In present-day cells, the rRNA genes have an important role in forming the nucleolus. In a diploid human cell, the rRNA genes are distributed into 10 clusters, located near the tips of five different chromosome pairs (see Figure 4–11). During interphase, these 10 chromosomes contribute DNA loops (containing the rRNA genes) to the nucleolus; in


Cell_Biology_Alberts

Cell_Biology_Alberts. M phase, when the chromosomes condense, the nucleolus fragments and then disappears. Then, in the telophase part of mitosis, as chromosomes return to their semi-dispersed state, the tips of the 10 chromosomes reform small nucleoli, which progressively coalesce into a single nucleolus (Figure 6–43 and Figure 6–44). As might be expected, the size of the nucleolus reflects the number of ribosomes that the cell is producing. Its size therefore varies greatly in different cells and can change in a single cell, occupying 25% of the total nuclear volume in cells that are making unusually large amounts of protein. Ribosome assembly is a complex process, the most important features of which are outlined in Figure 6–45. In addition to its central role in ribosome biogenesis, the nucleolus is the site where other noncoding RNAs are produced and other


Cell_Biology_Alberts

Cell_Biology_Alberts. RNA–protein complexes are assembled. For example, the U6 snRNP, which func tions in pre-mRNA splicing (see Figure 6–28), is composed of one RNA molecule and at least seven proteins. The U6 snRNA is chemically modified by snoRNAs in the nucleolus before its final assembly there into the U6 snRNP. Other important RNA–protein complexes, including telomerase (encountered in Chapter 5) and the signal-recognition particle (which we discuss in Chapter 12), are assembled at the nucleolus. Finally, the tRNAs (transfer RNAs) that carry the amino acids for protein synthesis are processed there as well; like the rRNA genes, the genes encoding tRNAs are clustered in the nucleolus. Thus, the nucleolus can be thought of as a large factory at which different noncoding RNAs are transcribed, processed, and assembled with proteins to form a large variety of ribonucleoprotein complexes.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–44 Nucleolar fusion. These light micrographs of human fibroblasts grown in culture show various stages of nucleolar fusion. After mitosis, each of the 10 human chromosomes that carry a cluster of rRNA genes begins to form a tiny nucleolus, but these rapidly coalesce as they grow to form the single large nucleolus typical of many interphase cells. (Courtesy of E.G. Jordan and J. McGovern.) The Nucleus Contains a Variety of Subnuclear Aggregates


Cell_Biology_Alberts

Cell_Biology_Alberts. Although the nucleolus is the most prominent structure in the nucleus, several other nuclear bodies have been observed and studied (Figure 6–46). These include Cajal bodies (named for the scientist who first described them in 1906) and interchromatin granule clusters (also called “speckles”). Like the nucleolus, these other nuclear structures lack membranes and are highly dynamic depending on the needs of the cell. Their assembly is likely mediated by the association of low complexity protein domains, as described in Chapter 3 (see Figure 3–36). Their appearance is the result of the tight association of protein and RNA components involved in the synthesis, assembly, and storage of macromolecules involved in gene expression. Cajal bodies are sites where the snRNPs and snoRNPs undergo their final maturation steps, and where the snRNPs are recycled and their RNAs are “reset” after the rearrangements that occur during splicing (see p. 321). In contrast, the interchromatin granule clusters


Cell_Biology_Alberts

Cell_Biology_Alberts. final maturation steps, and where the snRNPs are recycled and their RNAs are “reset” after the rearrangements that occur during splicing (see p. 321). In contrast, the interchromatin granule clusters have been proposed to be stockpiles of fully mature snRNPs and other RNA processing components that are ready to be used in the production of mRNA.


Cell_Biology_Alberts

Cell_Biology_Alberts. Scientists have had difficulties in working out the function of these small sub-nuclear structures, in part because their appearances can change dramatically as cells traverse the cell cycle or respond to changes in their environment. Moreover, Figure 6–45 The function of the nucleolus in ribosome and other ribonucleoprotein synthesis. The 45S precursor rRNA is packaged in a large ribonucleoprotein particle containing many ribosomal proteins imported from the cytoplasm. While this particle remains at the nucleolus, selected components are added and others discarded as it is processed into immature large and small ribosomal subunits. The two ribosomal subunits attain their final functional form only after each is individually transported through the nuclear pores into the cytoplasm. Other ribonucleoprotein complexes, including telomerase shown here, are also assembled in the nucleolus.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–46 Visualization of some prominent nuclear bodies. The protein fibrillarin (red), a component of several snoRNPs, is present at both nucleoli and Cajal bodies; the latter are indicated by the arrows. The Cajal bodies (but not the nucleoli) are also highlighted by staining one of their main components, the protein coilin; the superposition of the snoRNP and coilin stains appears pink. Interchromatin granule clusters (green) have been revealed by using antibodies against a protein involved in pre-mRNA splicing. DNA is stained blue by the dye DAPI. (From J.R. Swedlow and A.I. Lamond, Gen. Biol. 2:1–7, 2001. With permission from BioMed Central. Micrograph courtesy of Judith Sleeman.) disrupting a particular type of nuclear body often has little effect on cell viability. It seems that the main function of these aggregates is to bring components together at high concentration in order to speed up their assembly. For example, it is estimated that assembly of the U4/U6 snRNP (see


Cell_Biology_Alberts

Cell_Biology_Alberts. the main function of these aggregates is to bring components together at high concentration in order to speed up their assembly. For example, it is estimated that assembly of the U4/U6 snRNP (see Figure 6–28) occurs ten times more rapidly in Cajal bodies than would be the case if the same number of components were dispersed throughout the nucleus. Consequently, Cajal bodies appear dispensible in many types of cells but are absolutely required in situations where cells must proliferate rapidly, such as in early vertebrate development. Here, protein synthesis (which depends on RNA splicing) must be especially rapid, and delays can be lethal.


Cell_Biology_Alberts

Cell_Biology_Alberts. Given the prominence of nuclear bodies in RNA processing, it might be expected that pre-mRNA splicing would occur in a particular location in the nucleus, as it requires numerous RNA and protein components. However, as we have seen, the assembly of splicing components on pre-mRNA is co-transcriptional; thus, splicing must occur at many locations along chromosomes. Although a typical mammalian cell may be expressing on the order of 15,000 genes, transcription and RNA splicing takes place in only several thousand sites in the nucleus. These sites are highly dynamic and probably result from the association of transcription and splicing components to create small factories, the name given to specific aggregates containing a high local concentration of selected components that create biochemical assembly lines (Figure 6–47). Interchromatin tail of RNA polymerase


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–47 A model for an mRNA production factory. mRNA production is made more efficient in the nucleus by an aggregation of the many components needed for transcription and pre-mRNA processing, thereby producing a specialized biochemical factory. In (A), a postulated scaffold protein holds various components in the proximity of a transcribing RNA polymerase. Other key components are bound directly to the RNA polymerase tail, which likewise serves as a scaffold (see Figure 6–22), but for simplicity these are not shown here. In (B), a large number of such scaffolds have been brought together to form an aggregate that is highly enriched in the many components needed for the synthesis and processing of pre-mRNAs. Such a scaffold model can account for the several thousand sites of active RNA transcription and processing typically observed in the nucleus of a mammalian cell, each of which has a diameter of roughly 100nm and is estimated to contain, on average, about 10 RNA polymerase II


Cell_Biology_Alberts

Cell_Biology_Alberts. transcription and processing typically observed in the nucleus of a mammalian cell, each of which has a diameter of roughly 100nm and is estimated to contain, on average, about 10 RNA polymerase II molecules in addition to many other proteins. (C) Here, mRNA production factories and DNA replication factories have been visualized in the same mammalian cell by briefly incorporating differently modified nucleotides into each nucleic acid and detecting the RNA and DNA produced using antibodies, one (green) detecting the newly synthesized DNA and the other (red) detecting the newly synthesized RNA. (C, from D.G. Wansink et al., J. Cell Sci. 107:1449–1456, 1994. With permission from The Company of Biologists.) granule clusters—which contain stockpiles of RNA processing components—are often observed next to these sites of transcription, as though poised to replenish supplies. We can thus view the nucleus as organized into subdomains, with snRNPs, snoRNPs, and other nuclear components moving


Cell_Biology_Alberts

Cell_Biology_Alberts. next to these sites of transcription, as though poised to replenish supplies. We can thus view the nucleus as organized into subdomains, with snRNPs, snoRNPs, and other nuclear components moving among them in an orderly fashion according to the needs of the cell.


Cell_Biology_Alberts

Cell_Biology_Alberts. Before the synthesis of a particular protein can begin, the corresponding mRNA molecule must be produced by transcription. Bacteria contain a single type of RNA polymerase (the enzyme that carries out the transcription of DNA into RNA). An mRNA molecule is produced after this enzyme initiates transcription at a promoter, synthesizes the RNA by chain elongation, stops transcription at a terminator, and releases both the DNA template and the completed mRNA molecule. In eukaryotic cells, the process of transcription is much more complex, and there are three RNA polymerases—polymerase I, II, and III—that are related evolutionarily to one another and to the bacterial polymerase.


Cell_Biology_Alberts

Cell_Biology_Alberts. RNA polymerase II synthesizes eukaryotic mRNA. This enzyme requires a set of additional proteins, both the general transcription factors, and specific transcriptional activator proteins, to initiate transcription on a DNA template. It requires still more proteins (including chromatin remodeling complexes and histone-modifying enzymes) to initiate transcription on its chromatin templates inside the cell.


Cell_Biology_Alberts

Cell_Biology_Alberts. During the elongation phase of transcription, the nascent RNA undergoes three types of processing events: a special nucleotide is added to its 5ʹ end (capping), intron sequences are removed from the middle of the RNA molecule (splicing), and the 3ʹ end of the RNA is generated (cleavage and polyadenylation). Each of these processes is initiated by proteins that travel along with RNA polymerase II by binding to sites on its long, extended C-terminal tail. Splicing is unusual in that many of its key steps are carried out by specialized RNA molecules rather than proteins. Only properly processed mRNAs are passed through nuclear pore complexes into the cytosol, where they are translated into protein. For many genes, RNA, rather than protein, is the final product. In eukaryotes, these genes are usually transcribed by either RNA polymerase I or RNA polymerase


Cell_Biology_Alberts

Cell_Biology_Alberts. For many genes, RNA, rather than protein, is the final product. In eukaryotes, these genes are usually transcribed by either RNA polymerase I or RNA polymerase III. RNA polymerase I makes the ribosomal RNAs. After their synthesis as a large precursor, the rRNAs are chemically modified, cleaved, and assembled into the two ribosomal subunits in the nucleolus—a distinct subnuclear structure that also helps to process some smaller RNA–protein complexes in the cell. Additional subnuclear structures (including Cajal bodies and interchromatin granule clusters) are sites where components involved in RNA processing are assembled, stored, and recycled. The high concentration of components in such “factories” ensures that the processes being catalyzed are rapid and efficient.


Cell_Biology_Alberts

Cell_Biology_Alberts. In the preceding section, we have seen that the final product of some genes is an RNA molecule itself, such as the RNAs present in the snRNPs and in ribosomes. However, most genes in a cell produce mRNA molecules that serve as intermediaries on the pathway to proteins. In this section, we examine how the cell converts the information carried in an mRNA molecule into a protein molecule. This feat of translation was a strong focus of attention for biologists in the late 1950s, when it was posed as the “coding problem”: how is the information in a linear sequence of nucleotides in RNA translated into the linear sequence of a chemically quite different set of units—the amino acids in proteins? This fascinating question stimulated great excitement. Here was a cryptogram set up by nature that, after more than 3 billion years of evolution, could finally be solved by one of the products of evolution—human beings. And indeed, not only was the code cracked step by step, but in the year 2000 the


Cell_Biology_Alberts

Cell_Biology_Alberts. more than 3 billion years of evolution, could finally be solved by one of the products of evolution—human beings. And indeed, not only was the code cracked step by step, but in the year 2000 the structure of the elaborate machinery by which cells read this code—the ribosome—was finally revealed in atomic detail.


Cell_Biology_Alberts

Cell_Biology_Alberts. An mRNA Sequence Is Decoded in Sets of Three Nucleotides Once an mRNA has been produced by transcription and processing, the information present in its nucleotide sequence is used to synthesize a protein. Transcription is simple to understand as a means of information transfer: since DNA and RNA are chemically and structurally similar, the DNA can act as a direct template for the synthesis of RNA by complementary base-pairing. As the term transcription signifies, it is as if a message written out by hand is being converted, say, into a typewritten text. The language itself and the form of the message do not change, and the symbols used are closely related.


Cell_Biology_Alberts

Cell_Biology_Alberts. In contrast, the conversion of the information in RNA into protein represents a translation of the information into another language that uses quite different symbols. Moreover, since there are only 4 different nucleotides in mRNA and 20 different types of amino acids in a protein, this translation cannot be accounted for by a direct one-to-one correspondence between a nucleotide in RNA and an amino acid in protein. The nucleotide sequence of a gene, through the intermediary of mRNA, is instead translated into the amino acid sequence of a protein by rules that are known as the genetic code. This code was deciphered in the early 1960s.


Cell_Biology_Alberts

Cell_Biology_Alberts. The sequence of nucleotides in the mRNA molecule is read in consecutive groups of three. RNA is a linear polymer of four different nucleotides, so there are 4 × 4 × 4 = 64 possible combinations of three nucleotides: the triplets AAA, AUA, AUG, and so on. However, only 20 different amino acids are commonly found in proteins. Either some nucleotide triplets are never used, or the code is redundant and some amino acids are specified by more than one triplet. The second possibility is, in fact, the correct one, as shown by the completely deciphered genetic code in Figure 6–48. Each group of three consecutive nucleotides in RNA is called a codon, and each codon specifies either one amino acid or a stop to the translation process.


Cell_Biology_Alberts

Cell_Biology_Alberts. This genetic code is used universally in all present-day organisms. Although a few slight differences in the code have been found, these are chiefly in the DNA of mitochondria. Mitochondria have their own transcription and protein-synthesis systems that operate quite independently from those of the rest of the cell, and it is understandable that their tiny genomes have been able to accommodate minor changes to the code (discussed in Chapter 14). In principle, an RNA sequence can be translated in any one of three different reading frames, depending on where the decoding process begins (Figure 6–49). However, only one of the three possible reading frames in an mRNA encodes the required protein. We see later how a special punctuation signal at the beginning of each RNA message sets the correct reading frame at the start of protein synthesis. tRNA Molecules Match Amino Acids to Codons in mRNA


Cell_Biology_Alberts

Cell_Biology_Alberts. tRNA Molecules Match Amino Acids to Codons in mRNA The codons in an mRNA molecule do not directly recognize the amino acids they specify: the group of three nucleotides does not, for example, bind directly to the amino acid. Rather, the translation of mRNA into protein depends on adaptor molecules that can recognize and bind both to the codon and, at another site on their surface, to the amino acid. These adaptors consist of a set of small RNA molecules known as transfer RNAs (tRNAs), each about 80 nucleotides in length.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–48 The genetic code. The standard one-letter abbreviation for each amino acid is presented below its three-letter abbreviation (see Panel 3–1, pp. 112– 113, for the full name of each amino acid and its structure). By convention, codons are always written with the 5ʹ-terminal nucleotide to the left. Note that most amino acids are represented by more than one codon, and that there are some regularities in the set of codons that specifies each amino acid: codons for the same amino acid tend to contain the same nucleotides at the first and second positions, and vary at the third position. Three codons do not specify any amino acid but act as termination sites (stop codons), signaling the end of the protein-coding sequence. One codon—AUG—acts both as an initiation codon, signaling the start of a protein-coding message, and also as the codon that specifies methionine. Figure 6–49 The three possible reading frames in protein synthesis.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–49 The three possible reading frames in protein synthesis. In the process of translating a nucleotide sequence (blue) into an amino acid sequence (red), the sequence of nucleotides in an mRNA molecule is read from the 5ʹ end to the 3ʹ end in consecutive sets of three nucleotides. In principle, therefore, the same RNA sequence can specify three completely different amino acid sequences, depending on the reading frame. In reality, however, only one of these reading frames contains the actual message.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–50 A tRNA molecule. A tRNA specific for the amino acid phenylalanine (Phe) is depicted in various ways. (A) The cloverleaf structure showing the complementary base-pairing (red lines) that creates the double-helical regions of the molecule. The anticodon is the sequence of three nucleotides that base-pairs with a codon in mRNA. The amino acid matching the codon/anticodon pair is attached at the 3ʹ end of the tRNA. tRNAs contain some unusual bases, which are produced by chemical modification after the tRNA has been synthesized. For example, the bases denoted ψ (pseudouridine—see Figure 6–41) and D (dihydrouridine—see Figure 6–53) are derived from uracil. (B and C) Views of the L-shaped molecule, based on x-ray diffraction analysis. Although this diagram shows the tRNA for the amino acid phenylalanine, all other tRNAs have similar structures. (D) The tRNA icon we use in this book. (E) The linear nucleotide sequence of the molecule, color-coded to match (A), (B), and (C).


Cell_Biology_Alberts

Cell_Biology_Alberts. We saw earlier in this chapter that RNA molecules can fold into precise three-dimensional structures, and the tRNA molecules provide a striking example. Four short segments of the folded tRNA are double-helical, producing a molecule that looks like a cloverleaf when drawn schematically (Figure 6–50). For example, a 5ʹ-GCUC-3ʹ sequence in one part of a polynucleotide chain can form a relatively strong association with a 5ʹ-GAGC-3ʹ sequence in another region of the same molecule. The cloverleaf undergoes further folding to form a compact L-shaped structure that is held together by additional hydrogen bonds between different regions of the molecule (see Figure 6–50B and C).


Cell_Biology_Alberts

Cell_Biology_Alberts. Two regions of unpaired nucleotides situated at either end of the L-shaped molecule are crucial to the function of tRNA in protein synthesis. One of these regions forms the anticodon, a set of three consecutive nucleotides that pairs with the complementary codon in an mRNA molecule. The other is a short single-stranded region at the 3ʹend of the molecule; this is the site where the amino acid that matches the codon is attached to the tRNA.


Cell_Biology_Alberts

Cell_Biology_Alberts. We saw above that the genetic code is redundant; that is, several different codons can specify a single amino acid. This redundancy implies either that there is more than one tRNA for many of the amino acids or that some tRNA molecules can base-pair with more than one codon. In fact, both situations occur. Some amino acids have more than one tRNA and some tRNAs are constructed so that they require accurate base-pairing only at the first two positions of the codon and can tolerate a mismatch (or wobble) at the third position (Figure 6–51). This wobble base-pairing explains why so many of the alternative codons for an amino acid differ only in their third nucleotide (see Figure 6–48). In bacteria, wobble base-pairings make it possible to fit the 20 amino acids to their 61 codons with as


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–51 Wobble base-pairing between codons and anticodons. If the nucleotide listed in the first column is present at the third, or wobble, position of the codon, it can base-pair with any of the nucleotides listed in the second column. Thus, for example, when inosine (I) is present in the wobble position of the tRNA anticodon, the tRNA can recognize any one of three different codons in bacteria and either of two codons in eukaryotes. The inosine in tRNAs is formed from the deamination of adenosine (see Figure 6–53), a chemical modification that takes place after the tRNA has been synthesized. The nonstandard base pairs, including those made with inosine, are generally weaker than conventional base pairs. Codon–anticodon base-pairing is more stringent at positions 1 and 2 of the codon, where only conventional base pairs are permitted. The differences in wobble base-pairing interactions between bacteria and eukaryotes presumably result from subtle structural differences between


Cell_Biology_Alberts

Cell_Biology_Alberts. where only conventional base pairs are permitted. The differences in wobble base-pairing interactions between bacteria and eukaryotes presumably result from subtle structural differences between bacterial and eukaryotic ribosomes, the molecular machines that perform protein synthesis. (Adapted from C. Guthrie and


Cell_Biology_Alberts

Cell_Biology_Alberts. J. Abelson, in The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression, pp. 487–528. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 1982.) few as 31 kinds of tRNA molecules. The exact number of different kinds of tRNAs, however, differs from one species to the next. For example, humans have nearly 500 tRNA genes, and among them 48 different anticodons are represented. tRNAs Are Covalently Modified Before They Exit from the Nucleus


Cell_Biology_Alberts

Cell_Biology_Alberts. tRNAs Are Covalently Modified Before They Exit from the Nucleus Like most other eukaryotic RNAs, tRNAs are covalently modified before they are allowed to exit from the nucleus. Eukaryotic tRNAs are synthesized by RNA polymerase III. Both bacterial and eukaryotic tRNAs are typically synthesized as larger precursor tRNAs, which are then trimmed to produce the mature tRNA. In addition, some tRNA precursors (from both bacteria and eukaryotes) contain introns that must be spliced out. This splicing reaction differs chemically from pre-mRNA splicing; rather than generating a lariat intermediate, tRNA splicing uses a cutand-paste mechanism that is catalyzed by proteins (Figure 6–52). Trimming and splicing both require the precursor tRNA to be correctly folded in its cloverleaf configuration. Because misfolded tRNA precursors will not be processed properly, the trimming and splicing reactions serve as quality-control steps in the generation of tRNAs.


Cell_Biology_Alberts

Cell_Biology_Alberts. All tRNAs are modified chemically—nearly 1 in 10 nucleotides in each mature tRNA molecule is an altered version of a standard G, U, C, or A ribonucleotide. Over 50 different types of tRNA modifications are known; a few are shown in Figure 6–53. Some of the modified nucleotides—most notably inosine, produced by the deamination of adenosine—affect the conformation and base-pairing of the anticodon and thereby facilitate the recognition of the appropriate mRNA codon by the tRNA molecule (see Figure 6–51). Others affect the accuracy with which the tRNA is attached to the correct amino acid. Specific Enzymes Couple Each Amino Acid to Its Appropriate tRNA Molecule


Cell_Biology_Alberts

Cell_Biology_Alberts. We have seen that, to read the genetic code in DNA, cells make a series of different tRNAs. We now consider how each tRNA molecule becomes linked to the one amino acid in 20 that is its appropriate partner. Recognition and attachment of the correct amino acid depends on enzymes called aminoacyl-tRNA synthetases, which covalently couple each amino acid to its appropriate set of tRNA molecules (Figure 6–54 and Figure 6–55). Most cells have a different synthetase enzyme for each amino acid (that is, 20 synthetases in all); one attaches glycine to all tRNAs that recognize codons for glycine, another attaches alanine to all tRNAs that recognize codons for alanine, and so on. Many bacteria, however, have fewer than 20 synthetases, and the same synthetase enzyme is responsible for coupling more than one amino acid to the appropriate tRNAs. In these cases, a single synthetase places the identical amino acid on two different types of tRNAs, only one of which wobble codon possible base


Cell_Biology_Alberts

Cell_Biology_Alberts. more than one amino acid to the appropriate tRNAs. In these cases, a single synthetase places the identical amino acid on two different types of tRNAs, only one of which wobble codon possible base anticodon bases U A, G, or I C G or I A U or I G C or U wobble codon possible base anticodon bases U A, G, or I C G or I AU GC


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–52 Structure of a tRNA-splicing endonuclease docked to a precursor tRNA. The endonuclease (a four-subunit enzyme) removes the tRNA intron (dark blue, bottom). A second enzyme, a multifunctional tRNA ligase (not shown), then joins the two tRNA halves together. (Courtesy of Hong Li, Christopher Trotta, and John Abelson; PDB code: 2A9L.) ribose HHNNNNNCH3CH3ribose HNNOHHHHribose HNNSOHHtwo methyl groups added to G (N,N-dimethyl G) two hydrogens added to U (dihydro U) ribose HNNNNHHOdeamination of A (inosine) sulfur replaces oxygen in U (4-thiouridine) P P P P has an anticodon that matches the amino acid. A second enzyme then chemically modifies each “incorrectly” attached amino acid so that it now corresponds to the anticodon displayed by its covalently linked tRNA.


Cell_Biology_Alberts

Cell_Biology_Alberts. The synthetase-catalyzed reaction that attaches the amino acid to the 3ʹend of the tRNA is one of many reactions coupled to the energy-releasing hydrolysis of ATP (see pp. 64–65), and it produces a high-energy bond between the tRNA and the amino acid. The energy of this bond is used at a later stage in protein synthesis to link the amino acid covalently to the growing polypeptide chain. The aminoacyl-tRNA synthetase enzymes and the tRNAs are equally important in the decoding process (Figure 6–56). This was established by an experiment in


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–54 Amino acid activation by synthetase enzymes. An amino acid is activated for protein synthesis by an aminoacyl-tRNA synthetase enzyme in two steps. As indicated, the energy of ATP hydrolysis is used to attach each amino acid to its tRNA molecule in a high-energy linkage. The amino acid is first activated through the linkage of its carboxyl group directly to AMP, forming an adenylated amino acid; the linkage of the AMP, normally an unfavorable reaction, is driven by the hydrolysis of the ATP molecule that donates the AMP. Without leaving the synthetase enzyme, the AMP-linked carboxyl group on the amino acid is then transferred to a hydroxyl group on the sugar at the 3ʹ end of the tRNA molecule. This transfer joins the amino acid by an activated ester linkage to the tRNA and forms the final aminoacyl-tRNA molecule. The synthetase enzyme is not shown in this diagram. Figure 6–53 A few of the unusual nucleotides found in tRNA molecules.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–53 A few of the unusual nucleotides found in tRNA molecules. These nucleotides are produced by covalent modification of a normal nucleotide after it has been incorporated into an RNA chain. Two other types of modified nucleotides are shown in Figure 6–41. In most tRNA molecules, about 10% of the nucleotides are modified (see Figure 6–50). As shown in Figure 6–51, inosine is sometimes present at the wobble position in the tRNA anticodon.


Cell_Biology_Alberts

Cell_Biology_Alberts. which one amino acid (cysteine) was chemically converted into a different amino acid (alanine) after it already had been attached to its specific tRNA. When such “hybrid” aminoacyl-tRNA molecules were used for protein synthesis in a cell-free system, the wrong amino acid was inserted at every point in the protein chain where that tRNA was used. Although, as we shall see, cells have several quality control mechanisms to avoid this type of mishap, the experiment did establish that the genetic code is translated by two sets of adaptors that act sequentially. Each matches one molecular surface to another with great specificity, and it is their combined action that associates each sequence of three nucleotides in the mRNA molecule—that is, each codon—with its particular amino acid.


Cell_Biology_Alberts

Cell_Biology_Alberts. Several mechanisms working together ensure that an aminoacyl-tRNA synthetase links the correct amino acid to each tRNA. Most synthetase enzymes select the correct amino acid by a two-step mechanism. The correct amino acid has the highest affinity for the active-site pocket of its synthetase and is therefore favored over the other 19; in particular, amino acids larger than the correct one are excluded from the active site. However, accurate discrimination between two similar amino acids, such as isoleucine and valine (which differ by only a methyl linkage of amino acid to tRNA Figure 6–55 The structure of the aminoacyl-tRNA linkage. The carboxyl end of the amino acid forms an ester bond to ribose. Because the hydrolysis of this ester bond is associated with a large favorable change in free energy, an amino acid held in this way is said to be activated. Schematic drawing of the structure. The amino acid is linked to the nucleotide at the 3ʹ end of the tRNA (see Figure 6–50).


Cell_Biology_Alberts

Cell_Biology_Alberts. Schematic drawing of the structure. The amino acid is linked to the nucleotide at the 3ʹ end of the tRNA (see Figure 6–50). Actual structure corresponding to the boxed region in (A). There are two major classes of synthetase enzymes: one links the amino acid directly to the 3ʹ-OH group of the ribose, and the other links it initially to the 2ʹ-OH group. In the latter case, a subsequent transesterification reaction shifts the amino acid to the 3ʹ position. As in Figure 6–54, the “R group” indicates the side chain of the amino acid.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–56 The genetic code is translated by means of two adaptors that act one after another. The first adaptor is the aminoacyl-tRNA synthetase, which couples a particular amino acid to its corresponding tRNA; the second adaptor is the tRNA molecule itself, whose anticodon forms base pairs with the appropriate codon on the mRNA. An error in either step would cause the wrong amino acid to be incorporated into a protein chain (Movie 6.6). In the sequence of events shown, the amino acid tryptophan (Trp) is selected by the codon UGG on the mRNA.


Cell_Biology_Alberts

Cell_Biology_Alberts. group), is very difficult to achieve in a single step. A second discrimination step occurs after the amino acid has been covalently linked to AMP (see Figure 6–54): when tRNA binds, the synthetase tries to force the adenylated amino acid into a second editing pocket in the enzyme. The precise dimensions of this pocket exclude the correct amino acid, while allowing access by closely related amino acids. In the editing pocket, an amino acid is removed from the AMP (or from the tRNA itself if the aminoacyl-tRNA bond has already formed) by hydrolysis. This hydrolytic editing, which is analogous to the exonucleolytic proofreading by DNA polymerases, increases the overall accuracy of tRNA charging to approximately one mistake in 40,000 couplings (Figure 6–57).


Cell_Biology_Alberts

Cell_Biology_Alberts. The tRNA synthetase must also recognize the correct set of tRNAs, and extensive structural and chemical complementarity between the synthetase and the tRNA allows the synthetase to probe various features of the tRNA (Figure 6–58). Most tRNA synthetases directly recognize the matching tRNA anticodon; these synthetases contain three adjacent nucleotide-binding pockets, each of which is complementary in shape and charge to a nucleotide in the anticodon. For other synthetases, the nucleotide sequence of the amino acid-accepting arm (acceptor stem) is the key recognition determinant. In most cases, however, the synthetase “reads” the nucleotides at several different positions on the tRNA. Amino Acids Are Added to the C-terminal End of a Growing Polypeptide Chain


Cell_Biology_Alberts

Cell_Biology_Alberts. Amino Acids Are Added to the C-terminal End of a Growing Polypeptide Chain Having seen that each amino acid is first coupled to specific tRNA molecules, we now turn to the mechanism that joins these amino acids together to form proteins. The fundamental reaction of protein synthesis is the formation of a peptide bond between the carboxyl group at the end of a growing polypeptide chain and a free amino group on an incoming amino acid. Consequently, a protein is synthesized stepwise from its N-terminal end to its C-terminal end. Throughout the entire process, the growing carboxyl end of the polypeptide chain remains activated by its covalent attachment to a tRNA molecule (forming a peptidyl-tRNA). Each Figure 6–57 Hydrolytic editing. Aminoacyl tRNA synthetases correct their own coupling errors through hydrolytic editing of incorrectly attached amino acids. As described in the text, the correct amino acid is rejected by the editing site.


Cell_Biology_Alberts

Cell_Biology_Alberts. The error-correction process performed by DNA polymerase has similarities; however, it differs because the removal process depends strongly on a mispairing with the template (see Figure 5–8). (P, polymerization site; E, editing site.) Figure 6–58 The recognition of a tRNA molecule by its aminoacyl-tRNA synthetase. For this tRNA (tRNAGln), specific nucleotides in both the anticodon (dark blue) and the amino acid-accepting arm (green) allow the correct tRNA to be recognized by the synthetase enzyme (yellow-green). A bound ATP molecule is yellow. (Courtesy of Tom Steitz; PDB code: 1QRS.) peptidyl-tRNA attached to C-terminus of the growing polypeptide chain new peptidyl-tRNA molecule attached to C-terminus of the growing polypeptide chain


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–59 The incorporation of an amino acid into a protein. A polypeptide chain grows by the stepwise addition of amino acids to its C-terminal end. The formation of each peptide bond is energetically favorable because the growing C-terminus has been activated by the covalent attachment of a tRNA molecule. The peptidyl-tRNA linkage that activates the growing end is regenerated during each addition. The amino acid side chains have been abbreviated as R1, R2, R3, and R4; as a reference point, all of the atoms in the second amino acid in the polypeptide chain are shaded gray. The figure shows the addition of the fourth amino acid (red) to the growing chain.


Cell_Biology_Alberts

Cell_Biology_Alberts. addition disrupts this high-energy covalent linkage, but immediately replaces it with an identical linkage on the most recently added amino acid (Figure 6–59). In this way, each amino acid added carries with it the activation energy for the addition of the next amino acid rather than the energy for its own addition—an example of the “head growth” type of polymerization described in Figure 2–44. The RNA Message Is Decoded in Ribosomes


Cell_Biology_Alberts

Cell_Biology_Alberts. The RNA Message Is Decoded in Ribosomes The synthesis of proteins is guided by information carried by mRNA molecules. To maintain the correct reading frame and to ensure accuracy (about 1 mistake every 10,000 amino acids), protein synthesis is performed in the ribosome, a complex catalytic machine made from more than 50 different proteins (the ribosomal proteins) and several RNA molecules, the ribosomal RNAs (rRNAs). A typical eukaryotic cell contains millions of ribosomes in its cytoplasm (Figure 6–60). The large and small ribosome subunits are assembled at the nucleolus, where newly transcribed and modified rRNAs associate with the ribosomal proteins that have been transported into the nucleus after their synthesis in the cytoplasm. These two ribosomal subunits are then exported to the cytoplasm, where they join together to synthesize proteins.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–60 Ribosomes in the cytoplasm of a eukaryotic cell. This electron micrograph shows a thin section of a small region of cytoplasm. The ribosomes appear as black dots (red arrows). Some are free in the cytosol; others are attached to membranes of the endoplasmic reticulum. 400 nm (Courtesy of Daniel S. Friend.) 70S MW 2,800,000 30S (small) subunit MW 900,000 16S rRNA MW 4,200,000 60S (large) subunit 40S (small) subunit MW 1,400,000 28S rRNA 5.8S rRNA 18S rRNA MW 2,500,000 MW 1,600,000


Cell_Biology_Alberts

Cell_Biology_Alberts. MW 1,400,000 28S rRNA 5.8S rRNA 18S rRNA MW 2,500,000 MW 1,600,000 Figure 6–61 A comparison of bacterial and eukaryotic ribosomes. Despite differences in the number and size of their rRNA and protein components, both bacterial and eukaryotic ribosomes have nearly the same structure and they function similarly. Although the 18S and 28S rRNAs of the eukaryotic ribosome contain many nucleotides not present in their bacterial counterparts, these nucleotides are present as multiple insertions that form extra domains and leave the basic structure of the rRNA largely unchanged.


Cell_Biology_Alberts

Cell_Biology_Alberts. Eukaryotic and bacterial ribosomes have similar structures and functions, being composed of one large and one small subunit that fit together to form a complete ribosome with a mass of several million daltons (Figure 6–61). The small subunit provides the framework on which the tRNAs are accurately matched to the codons of the mRNA, while the large subunit catalyzes the formation of the peptide bonds that link the amino acids together into a polypeptide chain (see Figure 6–58).


Cell_Biology_Alberts

Cell_Biology_Alberts. When not actively synthesizing proteins, the two subunits of the ribosome are separate. They join together on an mRNA molecule, usually near its 5ʹ end, to initiate the synthesis of a protein. The mRNA is then pulled through the ribosome, three nucleotides at a time. As its codons enter the core of the ribosome, the mRNA nucleotide sequence is translated into an amino acid sequence using the tRNAs as adaptors to add each amino acid in the correct sequence to the growing end of the polypeptide chain. When a stop codon is encountered, the ribosome releases the finished protein, and its two subunits separate again. These subunits can then be used to start the synthesis of another protein on another mRNA molecule. Ribosomes operate with remarkable efficiency: in one second, a eukaryotic ribosome adds 2 amino acids to a polypeptide chain; the ribosomes of bacterial cells operate even faster, at a rate of about 20 amino acids per second.


Cell_Biology_Alberts

Cell_Biology_Alberts. To choreograph the many coordinated movements required for efficient translation, a ribosome contains four binding sites for RNA molecules: one is for the mRNA and three (called the A site, the P site, and the E site) are for tRNAs (Figure 6–62). A tRNA molecule is held tightly at the A and P sites only if its anticodon


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–62 The RNA-binding sites in the ribosome. Each ribosome has one binding site for mRNA and three binding sites for tRNA: the A, P, and E sites (short for aminoacyl-tRNA, peptidyl-tRNA, and exit, respectively). (A) A bacterial ribosome viewed with the small subunit in the front (dark green) and the large subunit in the back (light green). Both the rRNAs and the ribosomal proteins are illustrated. tRNAs are shown bound in the E site (red), the P site (orange), and the A site (yellow). Although all three tRNA sites are shown occupied here, during the process of protein synthesis not more than two of these sites are thought to contain tRNA molecules at any one time (see Figure 6–64). (B) Large and small ribosomal subunits arranged as though the ribosome in (A) were opened like a book. (C) The ribosome in (A) rotated through 90° and viewed with the large subunit on top and small subunit on the bottom. (D) Schematic representation of a ribosome [in the same orientation as (C)],


Cell_Biology_Alberts

Cell_Biology_Alberts. (C) The ribosome in (A) rotated through 90° and viewed with the large subunit on top and small subunit on the bottom. (D) Schematic representation of a ribosome [in the same orientation as (C)], which will be used in subsequent figures. (A, B, and C, adapted from M.M. Yusupov et al., Science 292:883–896, 2001. With permission from AAAS; courtesy of Albion Baucom and Harry Noller.) forms base pairs with a complementary codon (allowing for wobble) on the mRNA molecule that is threaded through the ribosome (Figure 6–63). The A and P sites are close enough together for their two tRNA molecules to be forced to form base pairs with adjacent codons on the mRNA molecule. This feature of the ribosome maintains the correct reading frame on the mRNA.


Cell_Biology_Alberts

Cell_Biology_Alberts. Once protein synthesis has been initiated, each new amino acid is added to the elongating chain in a cycle of reactions containing four major steps: tRNA binding (step 1), peptide bond formation (step 2), large subunit translocation (step 3), and small subunit translocation (step 4). As a result of the two translocation steps, the entire ribosome moves three nucleotides along the mRNA and is positioned to start the next cycle. Figure 6–64 illustrates this four-step process, beginning at a point at which three amino acids have already been linked together and there is a tRNA molecule in the P site on the ribosome, covalently joined to the C-terminal end of the short polypeptide. In step 1, a tRNA carrying the next amino acid in the chain binds to the ribosomal A site by forming base pairs with the mRNA codon positioned there, so that the P site and the A site contain adjacent bound tRNAs. In step 2, the carboxyl end of the polypeptide chain is released from the tRNA at the P site (by


Cell_Biology_Alberts

Cell_Biology_Alberts. with the mRNA codon positioned there, so that the P site and the A site contain adjacent bound tRNAs. In step 2, the carboxyl end of the polypeptide chain is released from the tRNA at the P site (by breakage of the high-energy bond between the tRNA and its amino acid) and joined to the free amino group of the amino acid linked to the tRNA at the A site, forming a new peptide bond. This central reaction of protein synthesis is catalyzed by a peptidyl transferase contained in the large ribosomal subunit. In step 3, the large subunit moves relative to the mRNA held by the small subunit, thereby shifting the acceptor stems of the two tRNAs to the E and P sites of the large subunit. In step 4, another series of conformational changes moves the small subunit and its bound mRNA exactly three nucleotides, ejecting the spent tRNA from the E site and resetting the ribosome so it is ready to receive the next aminoacyl-tRNA. Step 1 is then repeated with a new incoming aminoacyl-tRNA, and so on.


Cell_Biology_Alberts

Cell_Biology_Alberts. This four-step cycle is repeated each time an amino acid is added to the polypeptide chain, as the chain grows from its amino to its carboxyl end. The basic cycle of polypeptide elongation shown in outline in Figure 6–64 has an additional feature that makes translation especially efficient and accurate. Two elongation factors enter and leave the ribosome during each cycle, each hydrolyzing GTP to GDP and undergoing conformational changes in the process. These factors are called EF-Tu and EF-G in bacteria, and EF1 and EF2 in eukaryotes. Under some conditions in vitro, ribosomes can be forced to synthesize proteins


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–64 Translating an mRNA molecule. Each amino acid added to the growing end of a polypeptide chain is selected by complementary base-pairing between the anticodon on its attached tRNA molecule and the next codon on the mRNA chain. Because only one of the many types of tRNA molecules in a cell can base-pair with each codon, the codon determines the specific amino acid to be added to the growing polypeptide chain. The four-step cycle shown is repeated over and over during the synthesis of a protein. In step 1, an aminoacyl-tRNA molecule binds to a vacant A site on the ribosome. In step 2, a new peptide bond is formed. In step 3, the large subunit translocates relative to the small subunit, leaving the two tRNAs in hybrid sites: P on the large subunit and A on the small, for one; E on the large subunit and P on the small, for the other. In step 4, the small subunit translocates carrying its mRNA a distance of three nucleotides through the ribosome. This “resets” the ribosome with


Cell_Biology_Alberts

Cell_Biology_Alberts. the large subunit and P on the small, for the other. In step 4, the small subunit translocates carrying its mRNA a distance of three nucleotides through the ribosome. This “resets” the ribosome with a fully empty A site, ready for the next aminoacyl-tRNA molecule to bind. As indicated, the mRNA is translated in the 5ʹ-to-3ʹ direction, and the N-terminal end of a protein is made first, with each cycle adding one amino acid to the C-terminus of the polypeptide chain (Movie 6.7 and Movie 6.8).


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–63 The path of mRNA (blue) through the small ribosomal subunit. The orientation is the same as that in the right-hand panel of Figure 6–62B. (Courtesy of Harry F. Noller, based on data in G.Z. Yusupova et al., Cell 106:233–241, 2001. With permission from Elsevier.) Figure 6–65 Detailed view of the translation cycle. The outline of translation presented in Figure 6–64 has been expanded to show the roles of the two elongation factors EF-Tu and EF-G, which drive translation in the forward direction. As explained in the text, EF-Tu provides opportunities for proofreading of the codon–anticodon match. In this way, incorrectly paired tRNAs are selectively rejected, and the accuracy of translation is improved. The binding of a molecule of EF-G to the ribosome and the subsequent hydrolysis of GTP lead to a rearrangement of the ribosome structure, moving the mRNA being decoded exactly three nucleotides through it (Movie 6.9).


Cell_Biology_Alberts

Cell_Biology_Alberts. without the aid of these elongation factors and GTP hydrolysis, but this synthesis is very slow, inefficient, and inaccurate. Coupling the GTP hydrolysis-driven changes in the elongation factors to transitions between different states of the ribosome speeds up protein synthesis enormously. The cycles of elongation factor association, GTP hydrolysis, and dissociation also ensure that all such changes occur in the “forward” direction, helping translation to proceed efficiently (Figure 6–65).


Cell_Biology_Alberts

Cell_Biology_Alberts. In addition to moving translation forward, EF-Tu increases its accuracy. As we discussed in Chapter 3, EF-Tu can simultaneously bind GTP and aminoacyl-tR-NAs (see Figures 3–72 and 3–73), and it is in this form that the initial codon–anticodon interaction occurs in the A site of the ribosome. Because of the free-energy change associated with base-pair formation, a correct codon–anticodon match will bind more tightly than an incorrect interaction. However, this difference in affinity is relatively modest and cannot by itself account for the high accuracy of translation.


Cell_Biology_Alberts

Cell_Biology_Alberts. To increase the accuracy of this binding reaction, the ribosome and EF-Tu work together in the following ways. First, the 16s rRNA in the small subunit of the ribosome assesses the “correctness” of the codon–anticodon match by folding around it and probing its molecular details (Figure 6–66). When a correct match is found, the rRNA closes tightly around the codon–anticodon pair, causing a conformational change in the ribosome that triggers GTP hydrolysis by EF-Tu. Only when GTP is hydrolyzed does EF-Tu release its grip on the aminoacyl-tRNA and allow it to be used in protein synthesis. Incorrect codon–anticodon matches do not readily trigger this conformational change, and these errant tRNAs mostly fall off the ribosome before they can be used in protein synthesis. Proofreading, however, does not end here.


Cell_Biology_Alberts

Cell_Biology_Alberts. After GTP is hydrolyzed and EF-Tu dissociates from the ribosome, there is a second opportunity for the ribosome to prevent an incorrect amino acid from being added to the growing chain. There is a short time delay as the amino acid carried by the tRNA moves into position on the ribosome. This time delay is shorter for correct than incorrect codon–anticodon pairs. Moreover, incorrectly matched tRNAs dissociate more rapidly than those correctly bound because their interaction with the codon is weaker. Thus, most incorrectly bound tRNA molecules (as well as a significant number of correctly bound molecules) will leave the ribosome without being used for protein synthesis. The two proofreading steps, acting in series, are largely responsible for the 99.99% accuracy of the ribosome in translating RNA into protein.


Cell_Biology_Alberts

Cell_Biology_Alberts. Even if the wrong amino acid slips through the proofreading steps just described and is incorporated onto the growing polypeptide chain, there is still one more opportunity for the ribosome to detect the error and provide a solution, albeit one that is not, strictly speaking, proofreading. An incorrect codonanticodon interaction in the P site of the ribosome (which would occur after the misincorporation) causes an increased rate of misreading in the A site. Successive rounds of amino acid misincorporation eventually lead to premature termination of the protein by release factors, which are described below. Normally, these release factors act when translation of a protein is complete; here, they act early. Although this mechanism does not correct the original error, it releases the flawed protein for degradation, ensuring that no additional peptide synthesis is wasted on it.


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–66 Recognition of correct codon–anticodon matches by the small-subunit rRNA of the ribosome. Shown here is the interaction between a nucleotide of the small-subunit rRNA and the first nucleotide pair of a correctly paired codon–anticodon. Similar interactions form between other nucleotides of the rRNA and the second and third positions of codon– anticodon pair. The small-subunit rRNA can form this network of hydrogen bonds only when an anticodon is correctly matched to a codon. As explained in the text, this codon–anticodon monitoring by the small-subunit rRNA increases the accuracy of protein synthesis. (From J.M. Ogle et al., Science 292:897–902, 2001. With permission from AAAS.) Many Biological Processes Overcome the Inherent Limitations of Complementary Base-Pairing


Cell_Biology_Alberts

Cell_Biology_Alberts. Many Biological Processes Overcome the Inherent Limitations of Complementary Base-Pairing We have seen in this and the previous chapter that DNA replication, repair, transcription, and translation all rely on complementary base-pairing—G with C, and A with T (or U). However, if only the difference in hydrogen bonding is considered, a correct versus incorrect match should differ in affinity only by a factor of 10to 100-fold. These processes have an accuracy much higher than can be accounted for by this difference. Although the mechanisms used to “squeeze out” additional specificity from complementary base-pairing differ from one process to the next, two principles exemplified by the ribosome appear to be general.


Cell_Biology_Alberts

Cell_Biology_Alberts. The first is induced fit. We have seen that, before an amino acid is added to a growing polypeptide chain, the ribosome folds around the codon–anticodon interaction, and only when the match is correct is this folding completed and the reaction allowed to proceed. Thus, the codon–anticodon interaction is thereby checked twice—once by the initial complementary base-pairing and a second time by the folding of the ribosome, which depends on the correctness of the match. This same principle of induced fit is seen in transcription by RNA polymerase; here, an incoming nucleoside triphosphate initially forms a base pair with the template; at this point the enzyme folds around the base pair (thereby assessing its correctness) and, in doing so, creates the active site of the enzyme. The enzyme then covalently adds the nucleotide to the growing chain. Because their geometry is “wrong,” incorrect base pairs block this induced fit, and they are therefore likely to dissociate before being


Cell_Biology_Alberts

Cell_Biology_Alberts. then covalently adds the nucleotide to the growing chain. Because their geometry is “wrong,” incorrect base pairs block this induced fit, and they are therefore likely to dissociate before being incorporated into the growing chain.


Cell_Biology_Alberts

Cell_Biology_Alberts. A second principle used to increase the specificity of complementary base-pairing is called kinetic proofreading. We have seen that after the initial codon‒anticodon pairing and conformational change of the ribosome, GTP is hydrolyzed. This creates an irreversible step and starts the clock on a time delay during which the aminoacyl-tRNA moves into the proper position for catalysis. During this delay, those incorrect codon–anticodon pairs that have somehow slipped through the induced-fit scrutiny have a higher likelihood of dissociating than correct pairs. There are two reasons for this: (1) the interaction of the wrong tRNA with the codon is weaker, and (2) the delay is longer for incorrect than correct matches.


Cell_Biology_Alberts

Cell_Biology_Alberts. In its most general form, kinetic proofreading refers to a time delay that begins with an irreversible step such as ATP or GTP hydrolysis, during which an incorrect substrate is more likely to dissociate than a correct one. In this case, kinetic proofreading thus increases the specificity of complementary base-pairing above what is possible from simple thermodynamic associations alone. The increase in specificity produced by kinetic proofreading comes at an energetic cost in the form of ATP or GTP hydrolysis. Kinetic proofreading is believed to operate in many biological processes, but its role is understood particularly well for translation. Accuracy in Translation Requires an Expenditure of Free Energy


Cell_Biology_Alberts

Cell_Biology_Alberts. Accuracy in Translation Requires an Expenditure of Free Energy Translation by the ribosome is a compromise between the opposing constraints of accuracy and speed. We have seen, for example, that the accuracy of translation (1 mistake per 104 amino acids joined) requires time delays each time a new amino acid is added to a growing polypeptide chain, producing an overall speed of translation of 20 amino acids incorporated per second in bacteria. Mutant bacteria with a specific alteration in the small ribosomal subunit have longer delays and translate mRNA into protein with an accuracy considerably higher than this; however, protein synthesis is so slow in these mutants that the bacteria are barely able to survive.


Cell_Biology_Alberts

Cell_Biology_Alberts. We have also seen that attaining the observed accuracy of protein synthesis requires the expenditure of a great deal of free energy; this is expected, since, as discussed in Chapter 2, there is a price to be paid for any increase in order in the cell. In most cells, protein synthesis consumes more energy than any other biosynthetic process. At least four high-energy phosphate bonds are split to make each new peptide bond: two are consumed in charging a tRNA molecule with an amino acid (see Figure 6–54), and two more drive steps in the cycle of reactions occurring on the ribosome during protein synthesis itself (see Figure 6–65). In addition, extra energy is consumed each time that an incorrect amino acid linkage is hydrolyzed by a tRNA synthetase (see Figure 6–57) and each time that an incorrect tRNA enters the ribosome, triggers GTP hydrolysis, and is rejected (see Figure 6–65). To be effective, any proofreading mechanism must also allow an appreciable fraction of correct


Cell_Biology_Alberts

Cell_Biology_Alberts. that an incorrect tRNA enters the ribosome, triggers GTP hydrolysis, and is rejected (see Figure 6–65). To be effective, any proofreading mechanism must also allow an appreciable fraction of correct interactions to be removed; for this reason, proofreading is even more costly in energy than it might at first seem.


Cell_Biology_Alberts

Cell_Biology_Alberts. The Ribosome Is a Ribozyme The ribosome is a large complex composed of two-thirds RNA and one-third protein. The determination, in 2000, of the entire three-dimensional conformation of its large and small subunits is a major triumph of modern structural biology. The findings confirm earlier evidence that rRNAs—and not proteins—are responsible for the ribosome’s overall structure, its ability to position tRNAs on the mRNA, and its catalytic activity in forming covalent peptide bonds. The ribosomal RNAs are folded into highly compact, precise three-dimensional structures that form the compact core of the ribosome and determine its overall shape (Figure 6–67).


Cell_Biology_Alberts

Cell_Biology_Alberts. In marked contrast to the central positions of the rRNAs, the ribosomal proteins are generally located on the surface and fill in the gaps and crevices of the folded RNA (Figure 6–68). Some of these proteins send out extended regions of polypeptide chain that penetrate short distances into holes in the RNA core (Figure 6–69). The main role of the ribosomal proteins seems to be to stabilize the


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–67 Structure of the rRNAs in the large subunit of a bacterial ribosome, as determined by x-ray crystallography. (A) Three-dimensional conformations of the large-subunit rRNAs (5S and 23S) as they appear in the ribosome. One of the protein subunits of the ribosome (L1) is also shown as a reference point, since it forms a characteristic protrusion on the ribosome. (B) Schematic diagram of the secondary structure of the 23S rRNA, showing the extensive network of base-pairing. The structure has been divided into six “domains” whose colors correspond to those in (A). The secondary-structure diagram is highly schematized to represent as much of the structure as possible in two dimensions. To do this, several discontinuities in the RNA chain have been introduced, although in reality the 23S rRNA is a single RNA molecule. For example, the base of Domain III is continuous with the base of Domain IV even though a gap appears in the diagram. (Adapted from N. Ban et al., Science


Cell_Biology_Alberts

Cell_Biology_Alberts. the 23S rRNA is a single RNA molecule. For example, the base of Domain III is continuous with the base of Domain IV even though a gap appears in the diagram. (Adapted from N. Ban et al., Science 289:905–920, 2000. With permission from AAAS.)


Cell_Biology_Alberts

Cell_Biology_Alberts. RNA core, while permitting the changes in rRNA conformation that are necessary for this RNA to catalyze efficient protein synthesis. The proteins also aid in the initial assembly of the rRNAs that make up the core of the ribosome.


Cell_Biology_Alberts

Cell_Biology_Alberts. Not only are the A, P, and E binding sites for tRNAs formed primarily by ribosomal RNAs, but the catalytic site for peptide bond formation is also formed by RNA, as the nearest amino acid is located more than 1.8 nm away. This discovery came as a surprise to biologists because, unlike proteins, RNA does not contain easily ionizable functional groups that can be used to catalyze sophisticated reactions like peptide bond formation. Moreover, metal ions, which are often used by RNA molecules to catalyze chemical reactions (as discussed later in the chapter), were not observed at the active site of the ribosome. Instead, it is believed that the 23S rRNA forms a highly structured pocket that, through a network of hydrogen bonds, precisely orients the two reactants (the growing peptide chain and an aminoacyl-tRNA) and thereby greatly accelerates their covalent joining. An additional surprise came from the discovery that the tRNA in the P site contributes an important OH group to the active


Cell_Biology_Alberts

Cell_Biology_Alberts. an aminoacyl-tRNA) and thereby greatly accelerates their covalent joining. An additional surprise came from the discovery that the tRNA in the P site contributes an important OH group to the active site and participates directly in the catalysis. This mechanism may ensure that catalysis occurs only when the P site tRNA is properly positioned in the ribosome.


Cell_Biology_Alberts

Cell_Biology_Alberts. RNA molecules that possess catalytic activity are known as ribozymes. We saw earlier in this chapter that some ribozymes function in self-splicing reactions. In the final section of this chapter, we consider what the ability of RNA molecules to function as catalysts might mean for the early evolution of living cells. For now, we merely note that there is good reason to suspect that RNA rather than protein molecules served as the first catalysts for living cells. If so, the ribosome, with its RNA core, may be a relic of an earlier time in life’s history—when protein synthesis evolved in cells that were run almost entirely by ribozymes. Nucleotide Sequences in mRNA Signal Where to Start Protein Synthesis


Cell_Biology_Alberts

Cell_Biology_Alberts. Nucleotide Sequences in mRNA Signal Where to Start Protein Synthesis The initiation and termination of translation share features of the translation elongation cycle described above. The site at which protein synthesis begins on the mRNA is especially crucial, since it sets the reading frame for the whole length of the message. An error of one nucleotide either way at this stage would cause every subsequent codon in the message to be misread, resulting in a nonfunctional protein with a garbled sequence of amino acids. The initiation step is also important because for most genes it is the last point at which the cell can decide whether the mRNA is to be translated to produce a protein. The rate of this step is thus one determinant of the rate at which any particular protein will be synthesized. We shall see in Chapter 7 how regulation of this step occurs.


Cell_Biology_Alberts

Cell_Biology_Alberts. The translation of an mRNA begins with the codon AUG, and a special tRNA is required to start translation. This initiator tRNA always carries the amino acid methionine (in bacteria, a modified form of methionine—formylmethionine—is used), with the result that all newly made proteins have methionine as the first amino acid at their N-terminus, the end of a protein that is synthesized first. (This methionine is usually removed later by a specific protease.) The initiator tRNA is specially recognized by initiation factors because it has a nucleotide sequence distinct from that of the tRNA that normally carries methionine.


Cell_Biology_Alberts

Cell_Biology_Alberts. In eukaryotes, the initiator tRNA–methionine complex (Met–tRNAi) is first loaded into the small ribosomal subunit along with additional proteins called eukaryotic initiation factors, or eIFs. Of all the aminoacyl-tRNAs in the cell, only the methionine-charged initiator tRNA is capable of tightly binding the small ribosome subunit without the complete ribosome being present, and unlike other tRNAs it binds directly to the P site (Figure 6–70). Next, the small ribosomal subunit binds to the 5ʹ end of an mRNA molecule, which is recognized by virtue of its 5ʹ cap that has previously bound two initiation factors, eIF4E and eIF4G (see Figure 6–38). The small ribosomal subunit then moves forward (5ʹ to 3ʹ) along the mRNA, searching for the first AUG; additional initiation factors that act as ATP-powered


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–68 location of the protein components of the bacterial large ribosomal subunit. The rRNAs (5S and 23S) are shown in blue and the proteins of the large subunit in green. This view is toward the outside of the ribosome; the interface with the small subunit is on the opposite face. (PDB code: 1FFK.) Figure 6–69 Structure of the l15 protein in the large subunit of the bacterial ribosome. The globular domain of the protein lies on the surface of the ribosome and an extended region penetrates deeply into the RNA core of the ribosome. The L15 protein is shown in green and a portion of the ribosomal RNA core is shown in blue. (From D. Klein, P.B. Moore and T.A. Steitz, J. Mol. Biol. 340:141–177, 2004. With permission from Academic Press. PDB code: 1S72.)


Cell_Biology_Alberts

Cell_Biology_Alberts. T.A. Steitz, J. Mol. Biol. 340:141–177, 2004. With permission from Academic Press. PDB code: 1S72.) Figure 6–70 The initiation of protein synthesis in eukaryotes. Only three eIF2 of the many translation initiation factors required for this process are shown. Efficient translation initiation also requires the poly-A tail of the mRNA bound by poly-A-binding proteins, which, in turn, interact with eIF4G (see Figure 6–38). In this way, the translation apparatus ascertains that both ends of the mRNA are intact before initiating protein synthesis. Although only one GTP-hydrolysis event is shown in the figure, a second is known to occur subunit with just before the large and small ribosomal subunits join. In the last two steps initiator tRNA shown in the figure, the ribosome has begun the standard elongation cycle, bound to P site depicted in Figure 6–64. helicases facilitate this movement. In 90% of mRNAs, translation begins at the first


Cell_Biology_Alberts

Cell_Biology_Alberts. helicases facilitate this movement. In 90% of mRNAs, translation begins at the first AUG encountered by the small subunit. At this point, the initiation factors dis sociate, allowing the large ribosomal subunit to assemble with the complex and complete the ribosome. The initiator tRNA remains at the P site, leaving the A site vacant. Protein synthesis is therefore ready to begin (see Figure 6–70). The nucleotides immediately surrounding the start site in eukaryotic mRNAs influence the efficiency of AUG recognition during the above scanning process. If this recognition site differs substantially from the consensus recognition sequence (5ʹ-ACCAUGG-3ʹ), scanning ribosomal subunits will sometimes ignore the first


Cell_Biology_Alberts

Cell_Biology_Alberts. AUG codon in the mRNA and skip to the second or third AUG codon instead. Cells frequently use this phenomenon, known as “leaky scanning,” to produce two or more proteins, differing in their N-termini, from the same mRNA molecule. This mechanism allows some genes to produce the same protein with and without a signal sequence attached at its N-terminus, for example, so that the protein is directed to two different compartments in the cell. rial mRNAs have no 5ʹ caps to signal the ribosome where to begin searching for the start of translation. Instead, each bacterial mRNA contains a specific ribo- some-binding site (called the Shine–Dalgarno sequence, named after its discov-DISSOCIATE erers) that is located a few nucleotides upstream of the AUG at which translation


Cell_Biology_Alberts

Cell_Biology_Alberts. The mechanism for selecting a start codon in bacteria is different. Bacte is to begin. This nucleotide sequence, with the consensus 5ʹ-AGGAGGU-3ʹ, forms base pairs with the 16S rRNA of the small ribosomal subunit to position the initiating AUG codon in the ribosome. A set of translation initiation factors orchestrates this interaction, as well as the subsequent assembly of the large ribosomal subunit to complete the ribosome. Unlike a eukaryotic ribosome, a bacterial ribosome can readily assemble directly on a start codon that lies in the interior of an mRNA molecule, so long as a ribosome-binding site precedes it by several nucleotides. As a result, bac-AMINOACYL terial mRNAs are often polycistronic—that is, they encode several different pro teins, each of which is translated from the same mRNA molecule (Figure 6–71). In contrast, a eukaryotic mRNA generally encodes only a single protein, or more accurately, a single set of closely related proteins.


Cell_Biology_Alberts

Cell_Biology_Alberts. Stop Codons Mark the End of Translation The end of the protein-coding message is signaled by the presence of one of three stop codons (UAA, UAG, or UGA) (see Figure 6–48). These are not recognized by a tRNA and do not specify an amino acid, but instead signal to the ribosome to stop translation. Proteins known as release factors bind to any ribosome with a stop codon positioned in the A site, forcing the peptidyl transferase in the ribosome to catalyze the addition of a water molecule instead of an amino acid to the peptidyl-tRNA (Figure 6–72). This reaction frees the carboxyl end of the growing polypeptide chain from its attachment to a tRNA molecule, and since only this etc.


Cell_Biology_Alberts

Cell_Biology_Alberts. attachment normally holds the growing polypeptide to the ribosome, the com pleted protein chain is immediately released into the cytoplasm. The ribosome then releases its bound mRNA molecule and separates into the large and small subunits. These subunits can then assemble on this or another mRNA molecule to begin a new round of protein synthesis. Figure 6–71 Structure of a typical bacterial mRNA molecule. Unlike eukaryotic ribosomes, which typically require a capped 5ʹ end on the mRNA, prokaryotic ribosomes initiate translation at ribosome-binding sites (Shine–Dalgarno sequences), which can be located anywhere along an mRNA molecule. This property of their ribosomes permits bacteria to synthesize more than one type of protein from a single mRNA molecule.


Cell_Biology_Alberts

Cell_Biology_Alberts. During translation, the nascent polypeptide moves through a large, water-filled tunnel (approximately 10 nm × 1.5 nm) in the large subunit of the ribosome. The walls of this tunnel, made primarily of 23S rRNA, are a patchwork of tiny hydrophobic surfaces embedded in a more extensive hydrophilic surface. This structure is not complementary to any peptide, and thus provides a “Teflon” coating through which a polypeptide chain can easily slide. The dimensions of the tunnel suggest that nascent proteins are largely unstructured as they pass through the ribosome, although some α-helical regions of the protein can form before leaving the ribosome tunnel. As it leaves the ribosome, a newly synthesized protein must fold into its proper three-dimensional conformation to be useful to the cell. Later in this chapter we discuss how this folding occurs. First, however, we describe several additional aspects of the translation process itself.


Cell_Biology_Alberts

Cell_Biology_Alberts. The synthesis of most protein molecules takes between 20 seconds and several minutes. During this very short period, however, it is usual for multiple initiations to take place on each mRNA molecule being translated. As soon as the preceding ribosome has translated enough of the nucleotide sequence to move out of the way, the 5ʹ end of the mRNA is threaded into a new ribosome. The mRNA molecules being translated are therefore usually found in the form of polyribosomes (or polysomes): large cytoplasmic assemblies made up of several ribosomes spaced as close as 80 nucleotides apart along a single mRNA molecule (Figure 6–73). These multiple initiations allow the cell to make many more protein molecules in a given time than would be possible if each protein had to be completed before the next could start.


Cell_Biology_Alberts

Cell_Biology_Alberts. Both bacteria and eukaryotes use polysomes, and both employ additional strategies to speed up the overall rate of protein synthesis. Because bacterial mRNA does not need to be processed and is accessible to ribosomes while it is being made, ribosomes can attach to the free end of a bacterial mRNA molecule and start translating it even before the transcription of that RNA is complete, following closely behind the RNA polymerase as it moves along DNA. In eukaryotes, as we have seen, the 5ʹand 3ʹ ends of the mRNA interact (see Figure 6–73A); therefore, as soon as a ribosome dissociates, its two subunits are in an optimal position to reinitiate translation on the same mRNA molecule. There Are Minor Variations in the Standard Genetic Code


Cell_Biology_Alberts

Cell_Biology_Alberts. There Are Minor Variations in the Standard Genetic Code As discussed in Chapter 1, the genetic code (shown in Figure 6–48) applies to all three major branches of life, providing important evidence for the common ancestry of all life on Earth. Although rare, there are exceptions to this code. For example, Candida albicans, the most prevalent human fungal pathogen, translates the codon CUG as serine, whereas nearly all other organisms translate it as leucine. Mitochondria (which have their own genomes and encode much of their translational apparatus) often deviate from the standard code. For example, in mammalian mitochondria AUA is translated as methionine, whereas in the


Cell_Biology_Alberts

Cell_Biology_Alberts. Figure 6–72 The final phase of protein synthesis. The binding of a release factor to an A site bearing a stop codon terminates translation. The completed polypeptide is released and, in a series of reactions that requires additional proteins and GTP hydrolysis (not shown), the ribosome dissociates into its two separate subunits. messenger RNA Figure 6–73 A polyribosome. (A) Schematic drawing showing how a series of ribosomes can simultaneously translate the same eukaryotic mRNA molecule. (B) Electron micrograph of a polyribosome from a eukaryotic cell (Movie 6.10). (B, courtesy of John Heuser.) cytosol of the cell it is translated as isoleucine (see Table 14–3, p. 805). This type of deviation in the genetic code is “hardwired” into the organisms or the organelles in which it occurs.


Cell_Biology_Alberts

Cell_Biology_Alberts. A different type of variation, sometimes called translation recoding, occurs in many cells. In this case, other nucleotide sequence information present in an mRNA can change the meaning of the genetic code at a particular site in the mRNA molecule. The standard code allows cells to manufacture proteins using only 20 amino acids. However, bacteria, archaea, and eukaryotes have available to them a twenty-first amino acid that can be incorporated directly into a growing polypeptide chain through translation recoding. Selenocysteine, which is essential for the efficient function of a variety of enzymes, contains a selenium atom in place of the sulfur atom of cysteine. Selenocysteine is enzymatically produced from a serine attached to a special tRNA molecule that base-pairs with the UGA codon, a codon normally used to signal a translation stop. The mRNAs for proteins in which selenocysteine is to be inserted at a UGA codon carry an additional nearby nucleotide sequence in the mRNA that


Cell_Biology_Alberts

Cell_Biology_Alberts. a codon normally used to signal a translation stop. The mRNAs for proteins in which selenocysteine is to be inserted at a UGA codon carry an additional nearby nucleotide sequence in the mRNA that triggers this recoding event (Figure 6–74).


Cell_Biology_Alberts

Cell_Biology_Alberts. added to growing seryl-tRNA converted to selenocysteine selenocysteine tRNA signal that the preceding UGA encodes selenocysteine Figure 6–74 Incorporation of selenocysteine into a growing polypeptide chain. A specialized tRNA is charged with serine by the normal seryltRNA synthetase, and the serine is subsequently converted enzymatically to selenocysteine. A specific RNA structure in the mRNA (a stem and loop structure with a particular nucleotide sequence) signals that selenocysteine is to be inserted at the neighboring UGA codon. As indicated, this event requires the participation of a selenocysteine-specific translation factor. After the addition of selenocysteine, translation continues until a conventional stop codon is encountered. Inhibitors of Prokaryotic Protein Synthesis Are Useful as Antibiotics


Cell_Biology_Alberts

Cell_Biology_Alberts. Many of the most effective antibiotics used in modern medicine are compounds made by fungi that inhibit bacterial protein synthesis. Fungi and bacteria compete for many of the same environmental niches, and millions of years of coevolution have resulted in fungi producing potent bacterial inhibitors. Some of these drugs exploit the structural and functional differences between bacterial and eukaryotic ribosomes so as to interfere preferentially with the function of bacterial ribosomes. Thus, humans can take high dosages of some of these compounds without undue toxicity. Many antibiotics lodge in pockets in the ribosomal RNAs and simply interfere with the smooth operation of the ribosome; others block specific parts of the ribosome such as the exit channel (Figure 6–75). Table 6–4 lists some common antibiotics of this kind along with several other inhibitors of protein synthesis, some of which act on eukaryotic cells and therefore cannot be used as antibiotics.


Cell_Biology_Alberts

Cell_Biology_Alberts. Because they block specific steps in the processes that lead from DNA to protein, many of the compounds listed in Table 6–4 are useful for cell biological studies. Among the most commonly used drugs in such investigations are chloramphenicol, cycloheximide, and puromycin, all of which specifically inhibit protein synthesis. In a eukaryotic cell, for example, chloramphenicol inhibits protein synthesis on ribosomes only in mitochondria (and in chloroplasts in plants), presumably reflecting the prokaryotic origins of these organelles (discussed in Chapter 14). Cycloheximide, in contrast, affects only ribosomes in the cytosol. Puromycin is especially interesting because it is a structural analog of a tRNA molecule linked to an amino acid and is therefore another example of molecular mimicry; the ribosome mistakes it for an authentic amino acid and covalently incorporates it at the C-terminus of the growing peptide chain, thereby causing the premature termination and release of the


Cell_Biology_Alberts

Cell_Biology_Alberts. the ribosome mistakes it for an authentic amino acid and covalently incorporates it at the C-terminus of the growing peptide chain, thereby causing the premature termination and release of the polypeptide. As might be expected, puromycin inhibits protein synthesis in both prokaryotes and eukaryotes.