Biochemistry_Lippinco

Biochemistry_Lippinco. triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. As with replication, transcription is always in the 5′→3′ direction. In contrast to DNA pol, RNA pol does not require a primer and does not have a 3′→5′ exonuclease domain for proofreading. [Note: Misincorporation of a ribonucleotide causes RNA pol to pause, backtrack, cleave the transcript, and restart. Nonetheless, transcription has a higher error rate than does replication.] 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. Termination: The elongation of the single-stranded RNA chain continues until a termination signal is reached. Termination can be intrinsic (occur without additional proteins) or dependent upon the participation of a protein known as the ρ (rho) factor. a. ρ-Independent: Seen with most prokaryotic genes, this requires that a sequence in the DNA template generates a sequence in the nascent (newly made) RNA that is self-complementary (Fig. 31.9). This allows the RNA to fold back on itself, forming a GC-rich stem (stabilized by hydrogen bonds) plus a loop. This structure is known as a “hairpin.” Additionally, just beyond the hairpin, the RNA transcript contains a string of Us at the 3′-end. The bonding of these Us to the complementary As of the DNA template is weak. This facilitates the separation of the newly synthesized RNA from its DNA template, as the double helix “zips up” behind the RNA pol.


Biochemistry_Lippinco

Biochemistry_Lippinco. b. ρ-Dependent: This requires the participation of the additional protein rho, which is a hexameric ATPase with helicase activity. Rho binds a C-rich rho utilization (rut) site near the 5′-end of the nascent RNA and, using its ATPase activity, moves along the RNA until it reaches the RNA pol paused at the termination site. The ATP-dependent helicase activity of rho separates the RNA–DNA hybrid helix, causing the release of the RNA. 4. Antibiotics: Some antibiotics prevent bacterial cell growth by inhibiting RNA synthesis. For example, rifampin (rifampicin) inhibits transcription initiation by binding to the β subunit of prokaryotic RNA pol and preventing chain growth beyond three nucleotides (Fig. 31.10). Rifampin is important in the treatment of tuberculosis. Dactinomycin (actinomycin D) was the first antibiotic to find therapeutic application in tumor chemotherapy. It inserts (intercalates) between the DNA bases and inhibits transcription initiation and elongation.


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Biochemistry_Lippinco. D) was the first antibiotic to find therapeutic application in tumor chemotherapy. It inserts (intercalates) between the DNA bases and inhibits transcription initiation and elongation. IV. EUKARYOTIC GENE TRANSCRIPTION


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. EUKARYOTIC GENE TRANSCRIPTION The transcription of eukaryotic genes is a far more complicated process than transcription in prokaryotes. Eukaryotic transcription involves separate polymerases for the synthesis of rRNA, tRNA, and mRNA. In addition, a large number of proteins called transcription factors (TF) are involved. TF bind to distinct sites on the DNA within the core promoter region, close (proximal) to it, or some distance away (distal). They are required for both the assembly of a transcription initiation complex at the promoter and the determination of which genes are to be transcribed. [Note: Each eukaryotic RNA pol has its own promoters and TF that bind core promoter sequences.] For TF to recognize and bind to their specific DNA sequences, the chromatin structure in that region must be decondensed (relaxed) to allow access to the DNA. The role of transcription in the regulation of gene expression is discussed in Chapter 33. A. Chromatin structure and gene expression


Biochemistry_Lippinco

Biochemistry_Lippinco. The association of DNA with histones to form nucleosomes (see p. 425) affects the ability of the transcription machinery to access the DNA to be transcribed. Most actively transcribed genes are found in a relatively decondensed form of chromatin called euchromatin, whereas most inactive segments of DNA are found in highly condensed heterochromatin. The interconversion of these forms is called chromatin remodeling. A major component of chromatin remodeling is the covalent modification of histones (for example, the acetylation of lysine residues at the amino terminus of histone proteins), as shown in Figure 31.11. Acetylation, mediated by histone acetyltransferases (HAT), eliminates the positive charge on the lysine, thereby decreasing the interaction of the histone with the negatively charged DNA. Removal of the acetyl group by histone deacetylases (HDAC) restores the positive charge and fosters stronger interactions between histones and DNA. [Note: The ATP-dependent repositioning of


Biochemistry_Lippinco

Biochemistry_Lippinco. DNA. Removal of the acetyl group by histone deacetylases (HDAC) restores the positive charge and fosters stronger interactions between histones and DNA. [Note: The ATP-dependent repositioning of nucleosomes is also required to access DNA.]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Nuclear RNA polymerases There are three distinct types of RNA pol in the nucleus of eukaryotic cells. All are large enzymes with multiple subunits. Each type of RNA pol recognizes particular genes. [Note: Mitochondria contain a single RNA pol that resembles the bacterial enzyme.] 1. RNA polymerase I: This enzyme synthesizes the precursor of the 28S, 18S, and 5.8S rRNA in the nucleolus. 2. RNA polymerase II: This enzyme synthesizes the nuclear precursors of mRNA that are processed and then translated to proteins. RNA pol II also synthesizes certain small ncRNA, such as snoRNA, snRNA, and miRNA. a.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Promoters for RNA polymerase II: In some genes transcribed by RNA pol II, a sequence of nucleotides (TATAAA) that is nearly identical to that of the Pribnow box (see p. 436) is found centered ~25 nucleotides upstream of the transcription start site. This core promoter consensus sequence is called the TATA, or Hogness, box. In the majority of genes, however, no TATA box is present. Instead, different core promoter elements such as Inr (initiator) or DPE (downstream promoter element) are present (Fig. 31.12). [Note: No one consensus sequence is found in all core promoters.] Because these sequences are on the same molecule of DNA as the gene being transcribed, they are cis-acting. The sequences serve as binding sites for proteins known as general transcription factors (GTF), which in turn interact with each other and with RNA pol II. b.


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Biochemistry_Lippinco. General transcription factors: GTF are the minimal requirements for recognition of the promoter, recruitment of RNA pol II to the promoter, formation of the preinitiation complex, and initiation of transcription at a basal level (Fig. 31.13A). GTF are encoded by different genes, synthesized in the cytosol, and diffuse (transit) to their sites of action, and so are trans-acting. [Note: In contrast to the prokaryotic holoenzyme, eukaryotic RNA pol II does not itself recognize and bind the promoter. Instead, TFIID, a GTF containing TATA-binding protein and TATA-associated factors, recognizes and binds the TATA box (and other core promoter elements). TFIIF, another GTF, brings the polymerase to the promoter. The helicase activity of TFIIH melts the DNA, and its kinase activity phosphorylates polymerase, allowing it to clear the promoter.] c. Regulatory elements and transcriptional activators: Additional consensus sequences lie upstream of the core promoter (see Fig. 31.12). Those close to


Biochemistry_Lippinco

Biochemistry_Lippinco. allowing it to clear the promoter.] c. Regulatory elements and transcriptional activators: Additional consensus sequences lie upstream of the core promoter (see Fig. 31.12). Those close to the core promoter (within ~200 nucleotides) are the proximal regulatory elements, such as the CAAT and GC boxes. Those farther away are the distal regulatory elements such as enhancers (see d. below). Proteins known as transcriptional activators or specific transcription factors (STF) bind these regulatory elements. STF bind to promoter proximal elements to regulate the frequency of transcription initiation and to distal elements to mediate the response to signals such as hormones (see p. 472) and regulate which genes are expressed at a given point in time. A typical protein-coding eukaryotic gene has binding sites for many such factors. STF have two binding domains. One is a DNA-binding domain, the other is a transcription activation domain that recruits the GTF to the core promoter as well as


Biochemistry_Lippinco

Biochemistry_Lippinco. binding sites for many such factors. STF have two binding domains. One is a DNA-binding domain, the other is a transcription activation domain that recruits the GTF to the core promoter as well as coactivator proteins such as the HAT enzymes involved in chromatin modification. [Note: Mediator, a multisubunit coactivator of RNA pol II–catalyzed transcription, binds the polymerase, the GTF, and the STF and regulates transcription initiation.]


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Biochemistry_Lippinco. Transcriptional activators bind DNA through a variety of motifs, such as the helix-loop-helix, zinc finger, and leucine zipper (see p. 18).


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Biochemistry_Lippinco. d. Role of enhancers: Enhancers are special DNA sequences that increase the rate of initiation of transcription by RNA pol II. Enhancers are typically on the same chromosome as the gene whose transcription they stimulate (Fig. 31.13B). However, they can 1) be located upstream (to the 5′-side) or downstream (to the 3′-side) of the transcription start site, 2) be close to or thousands of base pairs away from the promoter (Fig. 31.14), and 3) occur on either strand of the DNA. Enhancers contain DNA sequences called response elements that bind STF. By bending or looping the DNA, STF can interact with other TF bound to a promoter and with RNA pol II, thereby stimulating transcription (see Fig. 31.13B). Mediator also binds enhancers. [Note: Although silencers are similar to enhancers in that they also can act over long distances, they reduce gene expression.] e. RNA polymerase II inhibitor: α-Amanitin, a potent toxin produced by the poisonous mushroom Amanita phalloides (sometimes called


Biochemistry_Lippinco

Biochemistry_Lippinco. they also can act over long distances, they reduce gene expression.] e. RNA polymerase II inhibitor: α-Amanitin, a potent toxin produced by the poisonous mushroom Amanita phalloides (sometimes called the “death cap”), binds RNA pol II tightly and slows its movement, thereby inhibiting mRNA synthesis.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. RNA polymerase III: This enzyme synthesizes tRNA, 5S rRNA, and some snRNA and snoRNA. V. POSTTRANSCRIPTIONAL MODIFICATION OF RNA A primary transcript is the initial, linear, RNA copy of a transcription unit (the segment of DNA between specific initiation and termination sequences). The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are posttranscriptionally modified by cleavage of the original transcripts by ribonucleases. tRNA are further modified to help give each species its unique identity. In contrast, prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic mRNA is extensively modified both co-and posttranscriptionally.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Ribosomal RNA rRNA of both prokaryotic and eukaryotic cells are generated from long precursor molecules called pre-rRNA. The 23S, 16S, and 5S rRNA of prokaryotes are produced from a single pre-rRNA molecule, as are the 28S, 18S, and 5.8S rRNA of eukaryotes (Fig. 31.15). [Note: Eukaryotic 5S rRNA is synthesized by RNA pol III and modified separately.] The prerRNA are cleaved by ribonucleases to yield intermediate-sized pieces of rRNA, which are further processed (trimmed by exonucleases and modified at some bases and riboses) to produce the required RNA species. [Note: In eukaryotes, rRNA genes are found in long, tandem arrays. rRNA synthesis and processing occur in the nucleolus, with base and sugar modifications facilitated by snoRNA.] B. Transfer RNA


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Transfer RNA Both eukaryotic and prokaryotic tRNA are also made from longer precursor molecules that must be modified (Fig. 31.16). Sequences at both ends of the molecule are removed, and, if present, an intron is removed from the anticodon loop by nucleases. Other posttranscriptional modifications include addition of a –CCA sequence by nucleotidyltransferase to the 3′terminal end of tRNA and modification of bases at specific positions to produce the unusual bases characteristic of tRNA (see p. 291). C. Eukaryotic messenger RNA The collection of all the primary transcripts synthesized in the nucleus by RNA pol II is known as heterogeneous nuclear RNA (hnRNA). The premRNA components of hnRNA undergo extensive co-and posttranscriptional modification in the nucleus and become mature mRNA. These modifications usually include the following. [Note: Pol II itself recruits the proteins required for the modifications.] 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. Addition of a 5′-cap: This is the first of the processing reactions for premRNA (Fig. 31.17). The cap is a 7-methylguanosine attached to the 5′terminal end of the mRNA through an unusual 5′→5′-triphosphate linkage that is resistant to most nucleases. Creation of the cap requires removal of the γ phosphoryl group from the 5′-triphosphate of the premRNA, followed by addition of guanosine monophosphate (from guanosine triphosphate) by the nuclear enzyme guanylyltransferase. Methylation of this terminal guanine occurs in the cytosol and is catalyzed by guanine-7-methyltransferase. S-Adenosylmethionine is the source of the methyl group (see p. 263). Additional methylation steps may occur. The addition of this 7-methylguanosine cap helps stabilize the mRNA and permits efficient initiation of translation (see p. 455). 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Addition of a 3′-poly-A tail: Most eukaryotic mRNA (with several exceptions, including those for the histones) have a chain of 40–250 adenylates (adenosine monophosphates) attached to the 3′-end (see Fig. 31.17). This poly-A tail is not transcribed from the DNA but rather is added by the nuclear enzyme, polyadenylate polymerase, using ATP as the substrate. The pre-mRNA is cleaved downstream of a consensus sequence, called the polyadenylation signal sequence (AAUAAA), found near the 3′-end of the RNA, and the poly-A tail is added to the new 3′end. Tailing terminates eukaryotic transcription. Tails help stabilize the mRNA, facilitate its exit from the nucleus, and aid in translation. After the mRNA enters the cytosol, the poly-A tail is gradually shortened. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Splicing: Maturation of eukaryotic mRNA usually involves removal from the primary transcript of RNA sequences (introns or intervening sequences) that do not code for protein. The remaining coding (expressed) sequences, the exons, are joined together to form the mature mRNA. The process of removing introns and joining exons is called splicing. The molecular complex that accomplishes these tasks is known as the spliceosome. A few eukaryotic primary transcripts contain no introns (for example, those from histone genes). Others contain a few introns, whereas some, such as the primary transcripts for the α chains of collagen, contain >50 introns that must be removed.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Role of small nuclear RNA: In association with multiple proteins, uracil-rich snRNA form five small nuclear ribonucleoprotein particles (snRNP, or “snurp”) designated as U1, U2, U4, U5, and U6 that mediate splicing. They facilitate the removal of introns by forming base pairs with the consensus sequences at each end of the intron (Fig. 31.18). [Note: In systemic lupus erythematosus, an autoimmune disease, patients produce antibodies against their own nuclear proteins such as snRNP.] b.


Biochemistry_Lippinco

Biochemistry_Lippinco. Mechanism: The binding of snRNP brings the sequences of neighboring exons into the correct alignment for splicing, allowing two transesterification reactions (catalyzed by the RNA of U2, U5, and U6) to occur. The 2′-OH group of an adenine nucleotide (known as the branch site A) in the intron attacks the phosphate at the 5′-end of the intron (splice-donor site), forming an unusual 2′→5′-phosphodiester bond and creating a “lariat” structure (see Fig. 31.18). The newly freed 3′-OH of exon 1 attacks the 5′-phosphate at the spliceacceptor site, forming a phosphodiester bond that joins exons 1 and 2. The excised intron is released as a lariat, which is typically degraded but may be a precursor for ncRNA such as snoRNA. [Note: The GU and AG sequences at the beginning and end, respectively, of introns are invariant. However, additional sequences are critical for splice-site recognition.] After introns have been removed and exons joined, the mature mRNA molecules pass into the cytosol through


Biochemistry_Lippinco

Biochemistry_Lippinco. are invariant. However, additional sequences are critical for splice-site recognition.] After introns have been removed and exons joined, the mature mRNA molecules pass into the cytosol through pores in the nuclear membrane. [Note: The introns in tRNA (see Fig. 31.16) are removed by a different mechanism.] c.


Biochemistry_Lippinco

Biochemistry_Lippinco. Effect of splice site mutations: Mutations at splice sites can lead to improper splicing and the production of aberrant proteins. It is estimated that at least 20% of all genetic diseases are a result of mutations that affect RNA splicing. For example, mutations that cause the incorrect splicing of β-globin mRNA are responsible for some cases of β-thalassemia, a disease in which the production of the βglobin protein is defective (see p. 38). Splice site mutations can result in exons being skipped (removed) or introns retained. They can also activate cryptic splice sites, which are sites that contain the 5′ or 3′ consensus sequence but are not normally used.


Biochemistry_Lippinco

Biochemistry_Lippinco. 4. Alternative splicing: The pre-mRNA molecules from >90% of human genes can be spliced in alternative ways in different tissues. Because this produces multiple variations of the mRNA and, therefore, of its protein product (Fig. 31.19), it is a mechanism for producing a large, diverse set of proteins from a limited set of genes. For example, the mRNA for tropomyosin (TM), an actin filament–binding protein of the cytoskeleton (and of the contractile apparatus in muscle cells), undergoes extensive tissue-specific alternative splicing with production of multiple isoforms of the TM protein. VI. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. Three major types of RNA participate in the process of protein synthesis: ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA), as shown in Figure 31.20. They are unbranched polymers of nucleotides but differ from DNA by containing ribose instead of deoxyribose and uracil instead of thymine. rRNA is a component of the ribosomes. tRNA serves as an adaptor molecule that carries a specific amino acid to the site of protein synthesis. mRNA (coding RNA) carries genetic information from DNA for use in protein synthesis. The process of RNA synthesis is called transcription, and the substrates are ribonucleoside triphosphates. The enzyme that synthesizes RNA is RNA polymerase (RNA pol). In prokaryotic cells, the core enzyme has five subunits (2 α, 1 β, 1 β′, and 1 Ω) and possesses 5′→3′ polymerase activity needed for transcription. The core enzyme requires an additional subunit, sigma (σ) factor, to recognize the nucleotide sequence (promoter region) at the beginning of the DNA


Biochemistry_Lippinco

Biochemistry_Lippinco. polymerase activity needed for transcription. The core enzyme requires an additional subunit, sigma (σ) factor, to recognize the nucleotide sequence (promoter region) at the beginning of the DNA to be transcribed. This region contains consensus sequences that are highly conserved and include the −10 Pribnow box and the −35 sequence. Another protein, rho (ρ), is required for termination of transcription of some genes. There are three distinct types of RNA pol in the nucleus of eukaryotic cells. RNA pol I synthesizes the precursor of rRNA in the nucleolus. In the nucleoplasm, RNA pol II synthesizes the precursors for mRNA and some noncoding RNA, and RNA pol III synthesizes the precursors of tRNA and 5S rRNA. In both prokaryotes and eukaryotes, RNA pol does not require a primer. Proofreading involves the polymerase backtracking and cleaving the transcript. Core promoters for genes transcribed by RNA pol II contain cis-acting consensus sequences, such as the TATA (Hogness) box, which


Biochemistry_Lippinco

Biochemistry_Lippinco. involves the polymerase backtracking and cleaving the transcript. Core promoters for genes transcribed by RNA pol II contain cis-acting consensus sequences, such as the TATA (Hogness) box, which serve as binding sites for transacting general transcription factors. Upstream of these are proximal regulatory elements, such as the CAAT and GC boxes, and distal regulatory elements, such as enhancers. Specific transcription factors (transcriptional activators) and Mediator complex bind these elements and regulate the frequency of transcription initiation, the response to signals such as hormones, and which genes are expressed at any given time. Eukaryotic transcription requires that the chromatin be relaxed (decondensed) in a process known as chromatin remodeling. A primary transcript is a linear copy of a transcription unit, the segment of DNA between specific initiation and termination sequences. The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are


Biochemistry_Lippinco

Biochemistry_Lippinco. is a linear copy of a transcription unit, the segment of DNA between specific initiation and termination sequences. The primary transcripts of both prokaryotic and eukaryotic tRNA and rRNA are posttranscriptionally modified. The rRNA are synthesized from long precursor molecules called pre-rRNA. These precursors are cleaved and trimmed by ribonucleases, producing the three largest rRNA, and bases and sugars are modified. Eukaryotic 5S rRNA is synthesized by RNA pol III and is modified separately. Prokaryotic and eukaryotic tRNA are also made from longer precursor molecules (pre-tRNA). If present, an intron is removed by nucleases, and both ends of the molecule are trimmed by ribonucleases. A 3′-CCA sequence is added, and bases at specific positions are modified. Prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic pre-mRNA is extensively modified co-and posttranscriptionally. For example, a 7-methylguanosine cap is attached to the 5′-end of the mRNA


Biochemistry_Lippinco

Biochemistry_Lippinco. identical to its primary transcript, whereas eukaryotic pre-mRNA is extensively modified co-and posttranscriptionally. For example, a 7-methylguanosine cap is attached to the 5′-end of the mRNA through a 5′→5′ linkage. A long poly-A tail, not transcribed from the DNA, is attached by polyadenylate polymerase to the 3′-end of most mRNA. Most eukaryotic mRNA also contains intervening sequences (introns) that must be removed for the mRNA to be functional. Their removal, as well as the joining of expressed sequences (exons), requires a spliceosome composed of small nuclear ribonucleoprotein particles (“snurps”) that mediate the process of splicing. Eukaryotic mRNA is monocistronic, containing information from just one gene, whereas prokaryotic mRNA is polycistronic.


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. 1.1. An 8-month-old male with severe anemia is found to have β-thalassemia. Genetic analysis shows that one of his β-globin genes has a mutation that creates a new splice-acceptor site 19 nucleotides upstream of the normal splice-acceptor site of the first intron. Which of the following best describes the new messenger RNA molecule that can be produced from this mutant gene? A. Exon 1 will be too short. B. Exon 1 will be too long. C. Exon 2 will be too short. D. Exon 2 will be too long. E. Exon 2 will be missing.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Exon 1 will be too short. B. Exon 1 will be too long. C. Exon 2 will be too short. D. Exon 2 will be too long. E. Exon 2 will be missing. Correct answer = D. Because the mutation creates an additional splice-acceptor site (the 3′-end) upstream of the normal acceptor site of intron 1, the 19 nucleotides that are usually found at the 3′-end of the excised intron 1 lariat can remain behind as part of exon 2. The presence of these extra nucleotides in the coding region of the mutant messenger RNA (mRNA) molecule will prevent the ribosome from translating the message into a normal β-globin protein molecule. Those mRNA for which the normal splice site is used to remove the first intron will be normal, and their translation will produce normal β-globin protein.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1.2. A 4-year-old child who easily tires and has trouble walking is diagnosed with Duchenne muscular dystrophy, an X-linked recessive disorder. Genetic analysis shows that the patient’s gene for the muscle protein dystrophin contains a mutation in its promoter region. Of the choices listed, which would be the most likely effect of this mutation? A. Initiation of dystrophin transcription will be defective. B. Termination of dystrophin transcription will be defective. C. Capping of dystrophin messenger RNA will be defective. D. Splicing of dystrophin messenger RNA will be defective. E. Tailing of dystrophin messenger RNA will be defective.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Capping of dystrophin messenger RNA will be defective. D. Splicing of dystrophin messenger RNA will be defective. E. Tailing of dystrophin messenger RNA will be defective. Correct answer = A. Mutations in the promoter typically prevent formation of the RNA polymerase II transcription initiation complex, resulting in a decrease in the initiation of messenger RNA (mRNA) synthesis. A deficiency of dystrophin mRNA will result in a deficiency in the production of the dystrophin protein. Capping, splicing, and tailing defects are not a consequence of promoter mutations. They can, however, result in mRNA with decreased stability (capping and tailing defects) or an mRNA in which exons have been skipped (lost) or introns retained (splicing defects). 1.3. A mutation to this sequence in eukaryotic messenger RNA (mRNA) will affect the process by which the 3′-end polyadenylate (poly-A) tail is added to the mRNA. A. AAUAAA B. CAAT C. CCA D. GU… A…AG E. TATAAA


Biochemistry_Lippinco

Biochemistry_Lippinco. A. AAUAAA B. CAAT C. CCA D. GU… A…AG E. TATAAA Correct answer = A. An endonuclease cleaves mRNA just downstream of this polyadenylation signal, creating a new 3′-end to which polyadenylate polymerase adds the poly-A tail using ATP as the substrate in a template-independent process. CAAT and TATAAA are sequences found in promoters for RNA polymerase II. CCA is added to the 3′-end of pre-transfer RNA by nucleotidyltransferase. GU…A…AG denotes an intron in eukaryotic premRNA. 1.4. This protein factor identifies the promoter of protein-coding genes in eukaryotes. A. Pribnow box B. Rho C. Sigma D. TFIID E. U1 Correct answer = D. The general transcription factor TFIID recognizes and binds core promoter elements such as the TATA-like box in eukaryotic protein-coding genes. These genes are transcribed by RNA polymerase II. The


Biochemistry_Lippinco

Biochemistry_Lippinco. Pribnow box is a cis-acting element in prokaryotic promoters. Rho is involved in the termination of prokaryotic transcription. Sigma is the subunit of prokaryotic RNA polymerase that recognizes and binds the prokaryotic promoter. U1 is a ribonucleoprotein involved in splicing of eukaryotic premRNA. 1.5. What is the sequence (conventionally written) of the RNA product of the DNA template sequence, GATCTAC, also conventionally written? Correct answer = 5′-GUAGAUC-3′. Nucleic acid sequences are conventionally written 5′ to 3′. The template strand (5′-GATCTAC-3′) is used as 3′CATCTAG-5′. The RNA product is complementary to the template strand (and identical to the coding strand), with U replacing T. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. Genetic information, stored in the chromosomes and transmitted to daughter cells through DNA replication, is expressed through transcription to RNA and, in the case of messenger RNA (mRNA), subsequent translation into proteins (polypeptides) as shown in Figure 32.1. [Note: The proteome is the complete set of proteins expressed in a cell.] The process of protein synthesis is called translation because the “language” of the nucleotide sequence on the mRNA is translated into the language of an amino acid sequence. Translation requires a genetic code, through which the information contained in the nucleotide sequence is expressed to produce a specific amino acid sequence. Any alteration in the nucleotide sequence may result in an incorrect amino acid being inserted into the protein, potentially causing disease or even death of the organism. Newly made immature (nascent) proteins undergo a number of processes to achieve their functional form. They must fold properly, and misfolding can


Biochemistry_Lippinco

Biochemistry_Lippinco. causing disease or even death of the organism. Newly made immature (nascent) proteins undergo a number of processes to achieve their functional form. They must fold properly, and misfolding can result in aggregation or degradation of the protein. Many proteins are covalently modified to alter their activities. Lastly, proteins are targeted to their final intra-or extracellular destinations by signals present in the proteins themselves.


Biochemistry_Lippinco

Biochemistry_Lippinco. II. THE GENETIC CODE The genetic code is a “dictionary” that identifies the correspondence between a sequence of nucleotide bases and a sequence of amino acids. Each individual “word” in the code is composed of three nucleotide bases. These genetic words are called codons. A. Codons Codons are presented in the mRNA language of adenine (A), guanine (G), cytosine (C), and uracil (U). Their nucleotide sequences are always written from the 5′-end to the 3′-end. The four nucleotide bases are used to produce the three-base codons. Therefore, 64 different combinations of bases exist, taken three at a time (a triplet code), as shown in the table in Figure 32.2. many common amino acids are shown as examples. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. many common amino acids are shown as examples. 1. How to translate a codon: This table can be used to translate any codon and, thus, to determine which amino acids are coded for by an mRNA sequence. For example, the codon AUG codes for methionine ([Met] see Fig. 32.2). [Note: AUG is the initiation (start) codon for translation.] Sixty-one of the 64 codons code for the 20 standard amino acids (see p. 1). 2. Termination codons: Three of the codons, UAA, UAG, and UGA, do not code for amino acids but, rather, are termination (also called stop, or nonsense) codons. When one of these codons appears in an mRNA sequence, synthesis of the polypeptide coded for by that mRNA stops. B. Characteristics


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Characteristics Usage of the genetic code is remarkably consistent throughout all living organisms. It is assumed that once the standard genetic code evolved in primitive organisms, any mutation (a permanent change in DNA sequence) that altered its meaning would have caused the alteration of most, if not all, protein sequences, resulting in lethality. Characteristics of the genetic code include the following. 1. Specificity: The genetic code is specific (unambiguous), because a particular codon always codes for the same amino acid. 2. Universality: The genetic code is virtually universal insofar as its specificity has been conserved from very early stages of evolution, with only slight differences in the manner in which the code is translated. [Note: An exception occurs in mitochondria, in which a few codons have meanings different than those shown in Figure 32.2. For example, UGA codes for tryptophan (Trp).] 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. Degeneracy: The genetic code is degenerate (sometimes called redundant). Although each codon corresponds to a single amino acid, a given amino acid may have more than one triplet coding for it. For example, arginine (Arg) is specified by six different codons (see Fig. 32.2). Only Met and Trp have just one coding triplet. 4. Nonoverlapping and commaless: The genetic code is nonoverlapping and commaless, meaning that the code is read from a fixed starting point as a continuous sequence of bases, taken three at a time without any punctuation between codons. For example, AGCUGGAUACAU is read as AGC UGG AUA CAU. C. Consequences of altering the nucleotide sequence Changing a single nucleotide base (a point mutation) in the coding region of an mRNA can lead to any one of three results (Fig. 32.3). 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Consequences of altering the nucleotide sequence Changing a single nucleotide base (a point mutation) in the coding region of an mRNA can lead to any one of three results (Fig. 32.3). 1. Silent mutation: The codon containing the changed base may code for the same amino acid. For example, if the serine (Ser) codon UCA is changed at the third base and becomes UCU, it still codes for Ser. This is termed a silent mutation. 2. Missense mutation: The codon containing the changed base may code for a different amino acid. For example, if the Ser codon UCA is changed at the first base and becomes CCA, it will code for a different amino acid (in this case, proline [Pro]). This is termed a missense mutation. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Nonsense mutation: The codon containing the changed base may become a termination codon. For example, if the Ser codon UCA is changed at the second base and becomes UAA, the new codon causes premature termination of translation at that point and the production of a shortened (truncated) protein. This is termed a nonsense mutation. [Note: The nonsense-mediated degradation pathway can degrade mRNA containing premature stops.] 4. Other mutations: These can alter the amount or structure of the protein produced by translation.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Trinucleotide repeat expansion: Occasionally, a sequence of three bases that is repeated in tandem will become amplified in number so that too many copies of the triplet occur. If this happens within the coding region of a gene, the protein will contain many extra copies of one amino acid. For example, expansion of the CAG codon in exon 1 of the gene for huntingtin protein leads to the insertion of many extra glutamine residues in the protein, causing the neurodegenerative disorder Huntington disease (Fig. 32.4). The additional glutamines result in an abnormally long protein that is cleaved, producing toxic fragments that aggregate in neurons. If the trinucleotide repeat expansion occurs in an untranslated region (UTR) of a gene, the result can be a decrease in the amount of protein produced, as seen in fragile X syndrome and myotonic dystrophy. Over 20 triplet expansion diseases are known. [Note: In fragile X syndrome, the most common cause of intellectual disability in males, the


Biochemistry_Lippinco

Biochemistry_Lippinco. as seen in fragile X syndrome and myotonic dystrophy. Over 20 triplet expansion diseases are known. [Note: In fragile X syndrome, the most common cause of intellectual disability in males, the expansion results in gene silencing through DNA hypermethylation (see p. 476).] b.


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Biochemistry_Lippinco. Splice site mutations: Mutations at splice sites (see p. 443) can alter the way in which introns are removed from pre-mRNA molecules, producing aberrant proteins. [Note: In myotonic dystrophy, a muscle disorder, gene silencing is the result of splicing alterations due to triplet expansion.] c.


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Biochemistry_Lippinco. Frameshift mutations: If one or two nucleotides are either deleted from or added to the coding region of an mRNA, a frameshift mutation occurs, altering the reading frame. This can result in a product with a radically different amino acid sequence or a truncated product due to the eventual creation of a termination codon (Fig. 32.5). If three nucleotides are added, a new amino acid is added to the peptide. If three are deleted, an amino acid is lost. Loss of three nucleotides maintains the reading frame but can result in serious pathology. For example, cystic fibrosis (CF), a chronic, progressive, inherited disease that primarily affects the pulmonary and digestive systems, is most commonly caused by deletion of three nucleotides from the coding region of a gene, resulting in the loss of phenylalanine (Phe, or F; see p. 5) at the 508th position (∆F508) in the CF transmembrane conductance regulator (CFTR) protein encoded by that gene. This ∆F508 mutation prevents normal folding of


Biochemistry_Lippinco

Biochemistry_Lippinco. of phenylalanine (Phe, or F; see p. 5) at the 508th position (∆F508) in the CF transmembrane conductance regulator (CFTR) protein encoded by that gene. This ∆F508 mutation prevents normal folding of CFTR, leading to its destruction by the proteasome (see p. 247). CFTR normally functions as a chloride channel in epithelial cells, and its loss results in the production of thick, sticky secretions in the lungs and pancreas, leading to lung damage and digestive deficiencies (see p. 174). The incidence of CF is highest (1 in 3,300) in those of Northern European origin. In >70% of individuals with CF, the ∆F508 mutation is the cause of the disease.


Biochemistry_Lippinco

Biochemistry_Lippinco. = guanine; U = uracil. III. COMPONENTS REQUIRED FOR TRANSLATION A large number of components are required for the synthesis of a protein. These include all the amino acids that are found in the finished product, the mRNA to be translated, transfer RNA (tRNA) for each of the amino acids, functional ribosomes, energy sources, and enzymes as well as noncatalytic protein factors needed for the initiation, elongation, and termination steps of polypeptide chain synthesis. A. Amino acids All the amino acids that eventually appear in the finished protein must be present at the time of protein synthesis. If one amino acid is missing, translation stops at the codon specifying that amino acid. [Note: This demonstrates the importance of having all the essential amino acids (see p. 262) in sufficient quantities in the diet to insure continued protein synthesis.] B. Transfer RNA


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Biochemistry_Lippinco. 262) in sufficient quantities in the diet to insure continued protein synthesis.] B. Transfer RNA At least one specific type of tRNA is required for each amino acid. In humans, there are at least 50 species of tRNA, whereas bacteria contain at least 30 species. Because there are only 20 different amino acids commonly carried by tRNA, some amino acids have more than one specific tRNA molecule. This is particularly true of those amino acids that are coded for by several codons.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Amino acid attachment site: Each tRNA molecule has an attachment site for a specific (cognate) amino acid at its 3′-end (Fig. 32.6). The carboxyl group of the amino acid is in an ester linkage with the 3′-hydroxyl of the ribose portion of the A nucleotide in the –CCA sequence at the 3′-end of the tRNA. [Note: A tRNA with a covalently attached (activated) amino acid is charged. Without an attached amino acid, it is uncharged.] 2. Anticodon: Each tRNA molecule also contains a three-base nucleotide sequence, the anticodon, which pairs with a specific codon on the mRNA (see Fig. 32.6). This codon specifies the insertion into the growing polypeptide chain of the amino acid carried by that tRNA. C. Aminoacyl-tRNA synthetases


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Biochemistry_Lippinco. This family of 20 different enzymes is required for attachment of amino acids to their corresponding tRNA. Each member of this family recognizes a specific amino acid and all the tRNA that correspond to that amino acid (isoaccepting tRNA, up to five per amino acid). Aminoacyl-tRNA synthetases catalyze a two-step reaction that results in the covalent attachment of the α-carboxyl group of an amino acid to the A in the –CCA sequence at the 3′-end of its corresponding tRNA. The overall reaction requires ATP, which is cleaved to adenosine monophosphate and inorganic pyrophosphate (PPi), as shown in Figure 32.7. The extreme specificity of the synthetases in recognizing both the amino acid and its cognate tRNA contributes to the high fidelity of translation of the genetic message. In addition to their synthetic activity, the aminoacyl-tRNA synthetases have a proofreading, or editing activity that can remove an incorrect amino acid from the enzyme or the tRNA molecule.


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Biochemistry_Lippinco. RNA (tRNA) by an aminoacyl-tRNA synthetase. PPi = pyrophosphate; Pi = monophosphate; ~ = high-energy bond. D. Messenger RNA The specific mRNA required as a template for the synthesis of the desired polypeptide must be present. [Note: In eukaryotes, mRNA is circularized for use in translation.] E. Functionally competent ribosomes


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Biochemistry_Lippinco. E. Functionally competent ribosomes As shown in Figure 32.8, ribosomes are large complexes of protein and ribosomal RNA (rRNA), in which rRNA predominates. They consist of two subunits (one large and one small) whose relative sizes are given in terms of their sedimentation coefficients, or S (Svedberg) values. [Note: Because the S values are determined by both shape and size, their numeric values are not strictly additive. For example, the prokaryotic 50S and 30S ribosomal subunits together form a 70S ribosome. The eukaryotic 60S and 40S subunits form an 80S ribosome.] Prokaryotic and eukaryotic ribosomes are similar in structure and serve the same function, namely, as the macromolecular complexes in which the synthesis of proteins occurs.


Biochemistry_Lippinco

Biochemistry_Lippinco. The small ribosomal subunit binds mRNA and determines the accuracy of translation by insuring correct base-pairing between the mRNA codon and the tRNA anticodon. The large ribosomal subunit catalyzes formation of the peptide bonds that link amino acid residues in a protein. 1. Ribosomal RNA: As discussed on p. 434, prokaryotic ribosomes contain three size species of rRNA, whereas eukaryotic ribosomes contain four (see Fig. 32.8). The rRNA are generated from a single pre-rRNA by the action of ribonucleases, and some bases and riboses are modified. 2. Ribosomal proteins: Ribosomal proteins are present in greater numbers in eukaryotic ribosomes than in prokaryotic ribosomes. These proteins play a variety of roles in the structure and function of the ribosome and its interactions with other components of the translation system. 3.


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Biochemistry_Lippinco. 3. A, P, and E sites: The ribosome has three binding sites for tRNA molecules: the A, P, and E sites, each of which extends over both subunits. Together, they cover three neighboring codons. During translation, the A site binds an incoming aminoacyl-tRNA as directed by the codon currently occupying this site. This codon specifies the next amino acid to be added to the growing peptide chain. The P site is occupied by peptidyl-tRNA. This tRNA carries the chain of amino acids that has already been synthesized. The E site is occupied by the empty tRNA as it is about to exit the ribosome. (See Fig. 32.13 for an illustration of the role of the A, P, and E sites in translation.) 4.


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Biochemistry_Lippinco. Cellular location: In eukaryotic cells, the ribosomes either are free in the cytosol or are in close association with the endoplasmic reticulum (which is then known as the rough endoplasmic reticulum, or RER). RER-associated ribosomes are responsible for synthesizing proteins (including glycoproteins; see p. 166) that are to be exported from the cell, incorporated into membranes, or imported into lysosomes (see p. 169 for an overview of the latter process). Cytosolic ribosomes synthesize proteins required in the cytosol itself or destined for the nucleus, mitochondria, or peroxisomes. [Note: Mitochondria contain their own ribosomes (55S) and their own unique, circular DNA. Most mitochondrial proteins, however, are encoded by nuclear DNA, synthesized completely in the cytosol, and then targeted to mitochondria.] F. Protein factors


Biochemistry_Lippinco

Biochemistry_Lippinco. F. Protein factors Initiation, elongation, and termination (or, release) factors are required for polypeptide synthesis. Some of these protein factors perform a catalytic function, whereas others appear to stabilize the synthetic machinery. [Note: A number of the factors are small, cytosolic G proteins and thus are active when bound to guanosine triphosphate (GTP) and inactive when bound to guanosine diphosphate (GDP). See p. 95 for a discussion of the membrane-associated G proteins.] G. Energy sources


Biochemistry_Lippinco

Biochemistry_Lippinco. G. Energy sources Cleavage of four high-energy bonds (see p. 73) is required for the addition of one amino acid to the growing polypeptide chain: two from ATP in the aminoacyl-tRNA synthetase reaction, one in the removal of PPi and one in the subsequent hydrolysis of the PPi, to two Pi by pyrophosphatase, and two from GTP, one for binding the aminoacyl-tRNA to the A site and one for the translocation step (see Fig. 32.13, p. 457). [Note: Additional ATP and GTP molecules are required for initiation in eukaryotes, whereas an additional GTP molecule is required for termination in both eukaryotes and prokaryotes.] Translation, then, is a major consumer of energy. IV. CODON RECOGNITION BY TRANSFER RNA Correct pairing of the codon in the mRNA with the anticodon of the tRNA is essential for accurate translation (see Fig. 32.6). Most tRNA (isoaccepting tRNA) recognize more than one codon for a given amino acid. A. Antiparallel binding between codon and anticodon


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Antiparallel binding between codon and anticodon Binding of the tRNA anticodon to the mRNA codon follows the rules of complementary and antiparallel binding, that is, the mRNA codon is read 5′→3′ by an anticodon pairing in the opposite (3′→5′) orientation (Fig. 32.9). [Note: Nucleotide sequences are always written in the 5′ to 3′ direction unless otherwise noted. Two nucleotide sequences orient in an antiparallel manner.] B. Wobble hypothesis


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Wobble hypothesis The mechanism by which a tRNA can recognize more than one codon for a specific amino acid is described by the wobble hypothesis, which states that codon–anticodon pairing follows the traditional Watson-Crick rules (G pairs with C and A pairs with U) for the first two bases of the codon but can be less stringent for the last base. The base at the 5′-end of the anticodon (the first base of the anticodon) is not as spatially defined as the other two bases. Movement of that first base allows nontraditional base-pairing with the 3′-base of the codon (the last base of the codon). This movement is called wobble and allows a single tRNA to recognize more than one codon. Examples of these flexible pairings are shown in Figure 32.9. The result of wobble is that 61 tRNA species are not required to read the 61 codons that code for amino acids. V. STEPS IN TRANSLATION


Biochemistry_Lippinco

Biochemistry_Lippinco. V. STEPS IN TRANSLATION The process of protein synthesis translates the 3-letter alphabet of nucleotide sequences on mRNA into the 20-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5′-end to its 3′-end, producing a protein synthesized from its amino (N)-terminal end to its carboxyl (C)-terminal end. Prokaryotic mRNA often have several coding regions (that is, they are polycistronic; see p. 434). Each coding region has its own initiation and termination codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA has only one coding region (that is, it is monocistronic). The process of translation is divided into three separate steps: initiation, elongation, and termination. Eukaryotic translation resembles that of prokaryotes in most aspects. Individual differences are noted in the text.


Biochemistry_Lippinco

Biochemistry_Lippinco. One important difference is that translation and transcription are temporally linked in prokaryotes, with translation starting before transcription is completed as a consequence of the lack of a nuclear membrane in prokaryotes. A. Initiation Initiation of protein synthesis involves the assembly of the components of the translation system before peptide-bond formation occurs. These components include the two ribosomal subunits, the mRNA to be translated, the aminoacyl-tRNA specified by the first codon in the message, GTP, and initiation factors that facilitate the assembly of this initiation complex (see Fig. 32.13). [Note: In prokaryotes, three initiation factors are known (IF-1, IF-2, and IF-3), whereas in eukaryotes, there are many (designated eIF to indicate eukaryotic origin). Eukaryotes also require ATP for initiation.] The following are two mechanisms by which the ribosome recognizes the nucleotide sequence (AUG) that initiates translation.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Shine-Dalgarno sequence: In Escherichia coli (E. coli), a purine-rich sequence of nucleotide bases, known as the Shine-Dalgarno (SD) sequence, is located six to ten bases upstream of the initiating AUG codon on the mRNA molecule (that is, near its 5′-end). The 16S rRNA component of the small (30S) ribosomal subunit has a nucleotide sequence near its 3′-end that is complementary to all or part of the SD sequence. Therefore, the 5′-end of the mRNA and the 3′-end of the 16S rRNA can form complementary base pairs, facilitating the positioning of the 30S subunit on the mRNA in close proximity to the initiating AUG codon (Fig. 32.10). 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. 5′-Cap: Eukaryotic mRNA do not have SD sequences. In eukaryotes, the small (40S) ribosomal subunit (aided by members of the eIF-4 family of proteins) binds close to the cap structure at the 5′-end of the mRNA and moves 5′→3′ along the mRNA until it encounters the initiator AUG. This scanning process requires ATP. Cap-independent initiation can occur if the 40S subunit binds to an internal ribosome entry site close to the start codon. [Note: Interactions between the cap-binding eIF-4 proteins and the poly-A tail–binding proteins on eukaryotic mRNA mediate circularization of the mRNA and likely prevent the use of incompletely processed mRNA in translation.] 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. Initiation codon: The initiating AUG is recognized by a special initiator tRNA (tRNAi). Recognition is facilitated by IF-2-GTP in prokaryotes and eIF-2-GTP (plus additional eIF) in eukaryotes. The charged tRNAi is the only tRNA recognized by (e)IF-2 and the only tRNA to go directly to the P site on the small subunit. [Note: Base modifications distinguish tRNAi from the tRNA used for internal AUG codons.] In bacteria and mitochondria, tRNAi carries an N-formylated methionine (fMet), as shown in Figure 32.11. After Met is attached to tRNAi, the formyl group is added by the enzyme transformylase, which uses N10-formyl tetrahydrofolate (see p. 267) as the carbon donor. In eukaryotes, tRNAi carries a Met that is not formylated. In both prokaryotic and eukaryotic cells, this N-terminal Met is usually removed before translation is completed. The large ribosomal subunit then joins the complex, and a functional ribosome is formed with the charged tRNAi in the P site. The A site is empty.


Biochemistry_Lippinco

Biochemistry_Lippinco. is usually removed before translation is completed. The large ribosomal subunit then joins the complex, and a functional ribosome is formed with the charged tRNAi in the P site. The A site is empty. [Note: Specific (e)IF function as anti-association factors and prevent premature addition of the large subunit.] The GTP on (e)IF-2 gets hydrolyzed to GDP. In eukaryotes, the guanine nucleotide exchange factor eIF-2B facilitates the reactivation of eIF-2-GDP through replacement of GDP by GTP.


Biochemistry_Lippinco

B. Elongation

Biochemistry_Lippinco. B. Elongation


Biochemistry_Lippinco

Biochemistry_Lippinco. Elongation of the polypeptide involves the addition of amino acids to the carboxyl end of the growing chain. Delivery of the aminoacyl-tRNA whose codon appears next on the mRNA template in the ribosomal A site (a process known as decoding) is facilitated in E. coli by elongation factors EF-Tu-GTP and EF-Ts and requires GTP hydrolysis. [Note: In eukaryotes, comparable elongation factors are EF-1α-GTP and EF-1βγ. Both EF-Ts and EF-1βγ function in guanine nucleotide exchange.] Peptide-bond formation between the α-carboxyl group of the amino acid in the P site and the αamino group of the amino acid in the A site is catalyzed by peptidyltransferase, an activity intrinsic to an rRNA of the large subunit (Fig. 32.12). [Note: Because this rRNA catalyzes the reaction, it is a ribozyme (see p. 54).] After the peptide bond has been formed, the peptide on the tRNA at the P site is transferred to the amino acid on the tRNA at the A site, a process known as transpeptidation. The ribosome then


Biochemistry_Lippinco

Biochemistry_Lippinco. 54).] After the peptide bond has been formed, the peptide on the tRNA at the P site is transferred to the amino acid on the tRNA at the A site, a process known as transpeptidation. The ribosome then advances three nucleotides toward the 3′-end of the mRNA. This process is known as translocation and, in prokaryotes, requires the participation of EF-G-GTP (eukaryotes use EF-2-GTP) and GTP hydrolysis. Translocation causes movement of the uncharged tRNA from the P to the E site for release and movement of the peptidyl-tRNA from the A to the P site. The process is repeated until a termination codon is encountered. [Note: Because of the length of most mRNA, more than one ribosome at a time can translate a message. Such a complex of one mRNA and a number of ribosomes is called a polysome, or polyribosome.]


Biochemistry_Lippinco

C. Termination

Biochemistry_Lippinco. C. Termination


Biochemistry_Lippinco

Biochemistry_Lippinco. Termination occurs when one of the three termination codons moves into the A site. These codons are recognized in E. coli by release factors: RF-1, which recognizes UAA and UAG, and RF-2, which recognizes UGA and UAA. The binding of these release factors results in hydrolysis of the bond linking the peptide to the tRNA at the P site, causing the nascent protein to be released from the ribosome. A third release factor, RF-3-GTP, then causes the release of RF-1 or RF-2 as GTP is hydrolyzed (see Fig. 32.13). [Note: Eukaryotes have a single release factor, eRF, which recognizes all three termination codons. A second factor, eRF-3, functions like the prokaryotic RF-3. See Figure 32.14 for a summary of the factors used in translation.] The steps in prokaryotic protein synthesis, as well as some antibiotic inhibitors of the process, are summarized in Figure 32.13. The newly synthesized polypeptide may undergo further modification as described below, and the ribosomal subunits, mRNA, tRNA,


Biochemistry_Lippinco

Biochemistry_Lippinco. antibiotic inhibitors of the process, are summarized in Figure 32.13. The newly synthesized polypeptide may undergo further modification as described below, and the ribosomal subunits, mRNA, tRNA, and protein factors can be recycled and used to synthesize another polypeptide. [Note: In prokaryotes, ribosome recycling factors mediate separation of the subunits. In eukaryotes, eRF and ATP hydrolysis are required.]


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Translation regulation Gene expression is most commonly regulated at the transcriptional level, but translation may also be regulated. An important mechanism by which this is achieved in eukaryotes is by covalent modification of eIF-2: Phosphorylated eIF-2 is inactive (see p. 476). In both eukaryotes and prokaryotes, regulation can also be achieved through proteins that bind mRNA and inhibit its use by blocking translation. E. Protein folding Proteins must fold to assume their functional, native state. Folding can be spontaneous (as a result of the primary structure) or facilitated by proteins known as chaperones (see p. 20). F. Protein targeting


Biochemistry_Lippinco

Biochemistry_Lippinco. Although most protein synthesis in eukaryotes is initiated in the cytoplasm, many proteins perform their functions within subcellular organelles or outside of the cell. Such proteins normally contain amino acid sequences that direct the proteins to their final locations. For example, secreted proteins are targeted during synthesis (cotranslational targeting) to the RER by the presence of an N-terminal hydrophobic signal sequence. The sequence is recognized by the signal recognition particle (SRP), a ribonucleoprotein that binds the ribosome, halts elongation, and delivers the ribosome–peptide complex to an RER membrane channel (the translocon) via interaction with the SRP receptor. Translation resumes, the protein enters the RER lumen, and its signal sequence is cleaved (Fig. 32.15). The protein moves through the RER and the Golgi, is processed, packaged into vesicles, and secreted. Proteins targeted after synthesis (posttranslational) include nuclear proteins that contain an


Biochemistry_Lippinco

Biochemistry_Lippinco. The protein moves through the RER and the Golgi, is processed, packaged into vesicles, and secreted. Proteins targeted after synthesis (posttranslational) include nuclear proteins that contain an internal, short, basic nuclear localization signal; mitochondrial matrix proteins that contain an N-terminal, amphipathic, α-helical mitochondrial entry sequence; and peroxisomal proteins that contain a C-terminal tripeptide signal.


Biochemistry_Lippinco

Biochemistry_Lippinco. VI. CO-AND POSTTRANSLATIONAL MODIFICATIONS Many polypeptides are covalently modified, either while they are still attached to the ribosome (cotranslational) or after their synthesis has been completed (posttranslational). These modifications may include removal of part of the translated sequence or the covalent addition of one or more chemical groups required for protein activity. A. Trimming Many proteins destined for secretion are initially made as large, precursor molecules that are not functionally active. Portions of the protein must be removed by specialized endoproteases, resulting in the release of an active molecule. The cellular site of the cleavage reaction depends on the protein to be modified. Some precursor proteins are cleaved in the RER or the Golgi; others are cleaved in developing secretory vesicles (for example, insulin; see Fig. 23.4, p. 309); and still others, such as collagen (see p. 47), are cleaved after secretion. B. Covalent attachments


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Covalent attachments Protein function can be affected by the covalent attachment of a variety of chemical groups (Fig. 32.16). Examples include the following. 1. Phosphorylation: Phosphorylation occurs on the hydroxyl groups of serine, threonine, or, less frequently, tyrosine residues in a protein. It is catalyzed by one of a family of protein kinases and may be reversed by the action of protein phosphatases. The phosphorylation may increase or decrease the functional activity of the protein. Several examples of phosphorylation reactions have been previously discussed (for example, see Chapter 11, p. 132, for the regulation of glycogen synthesis and degradation). 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Glycosylation: Many of the proteins that are destined to become part of a membrane or to be secreted from a cell have carbohydrate chains added en bloc to the amide nitrogen of an asparagine (N-linked) or built sequentially on the hydroxyl groups of a serine, threonine, or hydroxylysine (O-linked). N-glycosylation occurs in the RER and Oglycosylation in the Golgi. (The process of producing such glycoproteins was discussed on p. 165.) N-glycosylated acid hydrolases are targeted to the matrix of lysosomes by the phosphorylation of mannose residues at carbon 6 (see p. 169). 3. Hydroxylation: Proline and lysine residues of the α chains of collagen are extensively hydroxylated by vitamin C–dependent hydroxylases in the RER (see p. 47). 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Hydroxylation: Proline and lysine residues of the α chains of collagen are extensively hydroxylated by vitamin C–dependent hydroxylases in the RER (see p. 47). 4. Other covalent modifications: These may be required for the functional activity of a protein. For example, additional carboxyl groups can be added to glutamate residues by vitamin K–dependent carboxylation (see p. 393). The resulting γ-carboxyglutamate (Gla) residues are essential for the activity of several of the blood-clotting proteins. (See online Chapter 35.) Biotin is covalently bound to the ε-amino groups of lysine residues of biotin-dependent enzymes that catalyze carboxylation reactions such as pyruvate carboxylase (see Fig. 10.3 on p. 119). Attachment of lipids, such as farnesyl groups, can help anchor proteins to membranes (see p. 221). Many eukaryotic proteins are cotranslationally acetylated at the N-end. [Note: Reversible acetylation of histone proteins influences gene expression (see p. 476).]


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Protein degradation Proteins that are defective (for example, misfolded) or destined for rapid turnover are often marked for destruction by ubiquitination, the covalent attachment of chains of a small, highly conserved protein called ubiquitin (see Fig. 19.3 on p. 247). Proteins marked in this way are rapidly degraded by the proteasome, which is a macromolecular, ATP-dependent, proteolytic system located in the cytosol. For example, misfolding of the CFTR protein (see p. 450) results in its proteasomal degradation. [Note: If folding is impeded, unfolded proteins accumulate in the RER causing stress that triggers the unfolded protein response, in which the expression of chaperones is increased; global translation is decreased by eIF-2 phosphorylation; and the unfolded proteins are sent to the cytosol, ubiquitinated, and degraded in the proteasome by a process called ER-associated degradation.] VII. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. Codons are composed of three nucleotide bases presented in the messenger RNA (mRNA) language of adenine (A), guanine (G), cytosine (C), and uracil (U). They are always written 5′→3′. Of the 64 possible three-base combinations, 61 code for the 20 standard amino acids and 3 signal termination of protein synthesis (translation). Altering the nucleotide sequence in a codon can cause silent mutations (the altered codon codes for the original amino acid), missense mutations (the altered codon codes for a different amino acid), or nonsense mutations (the altered codon is a termination codon). Characteristics of the genetic code include specificity, universality, and degeneracy, and it is nonoverlapping and commaless (Fig. 32.17). Requirements for protein synthesis include all the amino acids that eventually appear in the finished protein; at least one specific type of transfer RNA (tRNA) for each amino acid; one aminoacyl-tRNA synthetase for each amino acid; the mRNA coding for the protein


Biochemistry_Lippinco

Biochemistry_Lippinco. eventually appear in the finished protein; at least one specific type of transfer RNA (tRNA) for each amino acid; one aminoacyl-tRNA synthetase for each amino acid; the mRNA coding for the protein to be synthesized; fully competent ribosomes (70S in prokaryotes, 80S in eukaryotes); protein factors needed for initiation, elongation, and termination of protein synthesis; and ATP and guanosine triphosphate (GTP) as energy sources. tRNA has an attachment site for a specific amino acid at its 3′-end and an anticodon region that can recognize the codon specifying the amino acid the tRNA is carrying. Ribosomes are large complexes of protein and ribosomal RNA (rRNA). They consist of two subunits, 30S and 50S in prokaryotes and 40S and 60S in eukaryotes. Each ribosome has three binding sites for tRNA molecules: the A, P, and E sites that cover three neighboring codons. The A site binds an incoming aminoacyl-tRNA, the P site is occupied by peptidyl-tRNA, and the E site is occupied by the empty


Biochemistry_Lippinco

Biochemistry_Lippinco. molecules: the A, P, and E sites that cover three neighboring codons. The A site binds an incoming aminoacyl-tRNA, the P site is occupied by peptidyl-tRNA, and the E site is occupied by the empty tRNA as it is about to exit the ribosome. Recognition of an mRNA codon is accomplished by the tRNA anticodon, which binds to the codon following the rules of complementarity and antiparallel binding. The wobble hypothesis states that the first (5′) base of the anticodon is not as spatially defined as the other two bases. Movement of that first base allows nontraditional base-pairing with the last (3′) base of the codon, thus allowing a single tRNA to recognize more than one codon for a specific amino acid. For initiation of protein synthesis, the components of the translation system are assembled, and mRNA associates with the small ribosomal subunit. The process requires initiation factors (IF). In prokaryotes, a purine-rich region of the mRNA (the Shine-Dalgarno sequence) base-pairs with a


Biochemistry_Lippinco

Biochemistry_Lippinco. and mRNA associates with the small ribosomal subunit. The process requires initiation factors (IF). In prokaryotes, a purine-rich region of the mRNA (the Shine-Dalgarno sequence) base-pairs with a complementary sequence on 16S rRNA, resulting in the positioning of the small subunit on the mRNA so that translation can begin. The 5′-cap (bound by proteins of the eIF-4 family) on eukaryotic mRNA is used to position the small subunit on the mRNA. The initiation codon is AUG, and N-formylmethionine is the initiating amino acid in prokaryotes, whereas methionine is used in eukaryotes. The charged initiating tRNA (tRNAi) is brought to the P site by (e)IF-2. In elongation, the polypeptide chain is lengthened by the addition of amino acids to the carboxyl end of its growing chain. The process requires elongation factors that facilitate the binding of the aminoacyl-tRNA to the A site as well as the movement of the ribosome along the mRNA. The formation of the peptide bond is catalyzed by


Biochemistry_Lippinco

Biochemistry_Lippinco. requires elongation factors that facilitate the binding of the aminoacyl-tRNA to the A site as well as the movement of the ribosome along the mRNA. The formation of the peptide bond is catalyzed by peptidyltransferase, which is an activity intrinsic to the rRNA of the large subunit and, therefore, is a ribozyme. Following peptide-bond formation, the ribosome advances along the mRNA in the 5′→3′ direction to the next codon (translocation). Because of the length of most mRNA, more than one ribosome at a time can translate a message, forming a polysome. Termination begins when one of the three termination codons moves into the A site. These codons are recognized by release factors. The newly synthesized protein is released from the ribosomal complex, and the ribosome is dissociated from the mRNA. Initiation, elongation, and termination are driven by the hydrolysis of GTP. Initiation in eukaryotes also requires ATP for scanning. Numerous antibiotics interfere with the process of protein


Biochemistry_Lippinco

Biochemistry_Lippinco. mRNA. Initiation, elongation, and termination are driven by the hydrolysis of GTP. Initiation in eukaryotes also requires ATP for scanning. Numerous antibiotics interfere with the process of protein synthesis. Many polypeptide chains are covalently modified during or after translation. Such modifications include amino acid removal; phosphorylation, which may activate or inactivate the protein; glycosylation, which plays a role in protein targeting; and hydroxylation such as that seen in collagen. Protein targeting can be either cotranslational (as with secreted proteins) or posttranslational (as with mitochondrial matrix proteins). Proteins must fold to achieve their functional form. Folding can be spontaneous or facilitated by chaperones. Proteins that are defective (for example, misfolded) or destined for rapid turnover are marked for destruction by the attachment of chains of a small, highly conserved protein called ubiquitin. Ubiquitinated proteins are rapidly degraded by a


Biochemistry_Lippinco

Biochemistry_Lippinco. or destined for rapid turnover are marked for destruction by the attachment of chains of a small, highly conserved protein called ubiquitin. Ubiquitinated proteins are rapidly degraded by a cytosolic complex known as the proteasome.


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. 2.1. A 20-year-old man with a microcytic anemia is found to have an abnormal form of β-globin (Hemoglobin Constant Spring) that is 172 amino acids long, rather than the 141 found in the normal protein. Which of the following point mutations is consistent with this abnormality? Use Figure 32.2 to answer the question. A. CGA→UGA B. GAU→GAC C. GCA→GAA D. UAA→CAA D. UAA→UAG


Biochemistry_Lippinco

Biochemistry_Lippinco. A. CGA→UGA B. GAU→GAC C. GCA→GAA D. UAA→CAA D. UAA→UAG Correct answer = D. Mutating the normal termination (stop) codon from UAA to CAA in β-globin messenger RNA causes the ribosome to insert a glutamine at that point. It will continue extending the protein chain until it comes upon the next stop codon farther down the message, resulting in an abnormally long protein. The replacement of CGA (arginine) with UGA (stop) would cause the protein to be too short. GAU and GAC both code for aspartate and would cause no change in the protein. Changing GCA (alanine) to GAA (glutamate) would not change the size of the protein product. A change from UAA to UAG would simply change one termination codon for another and would have no effect on the protein.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2.2. A pharmaceutical company is studying a new antibiotic that inhibits bacterial protein synthesis. When this antibiotic is added to an in vitro protein synthesis system that is translating the messenger RNA sequence AUGUUUUUUUAG, the only product formed is the dipeptide fMet-Phe. What step in protein synthesis is most likely inhibited by the antibiotic? A. Initiation B. Binding of a charged transfer RNA to the ribosomal A site C. Peptidyltransferase activity D. Ribosomal translocation E. Termination Correct answer = D. Because fMet-Phe (formylated methionyl-phenylalanine) is made, the ribosomes must be able to complete initiation, bind Phe-tRNA to the A site, and use peptidyltransferase activity to form the first peptide bond. Because the ribosome is not able to proceed any further, ribosomal movement (translocation) is most likely the inhibited step. Therefore, the ribosome is stopped before it reaches the termination codon of this message.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2.3. A transfer RNA (tRNA) molecule that is supposed to carry cysteine (tRNAcys) is mischarged, so that it actually carries alanine (ala-tRNAcys). Assuming no correction occurs, what will be the fate of this alanine residue during protein synthesis? It will: A. be incorporated into a protein in response to a codon for alanine. B. be incorporated into a protein in response to a codon for cysteine. C. be incorporated randomly at any codon. D. remain attached to the tRNA because it cannot be used for protein synthesis. E. be chemically converted to cysteine by cellular enzymes. Correct answer = B. Once an amino acid is attached to a tRNA molecule, only the anticodon of that tRNA determines the specificity of incorporation. Therefore, the incorrectly activated alanine will be incorporated into the protein at a position determined by a cysteine codon.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2.4. In a patient with cystic fibrosis (CF) caused by the ∆F508 mutation, the mutant CF transmembrane conductance regulator (CFTR) protein folds incorrectly. The patient’s cells modify this abnormal protein by attaching ubiquitin molecules to it. What is the fate of this modified CFTR protein? A. It performs its normal function because the ubiquitin largely corrects for the effect of the mutation. B. It is degraded by the proteasome. C. It is placed into storage vesicles. D. It is repaired by cellular enzymes. E. It is secreted from the cell. Correct answer = B. Ubiquitination usually marks old, damaged, or misfolded proteins for destruction by the cytosolic proteasome. There is no known cellular mechanism for repair of damaged proteins. 2.5. Many antimicrobials inhibit translation. Which of the following antimicrobials is correctly paired with its mechanism of action? A. Erythromycin binds to the 60S ribosomal subunit. B. Puromycin inactivates elongation factor-2.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Erythromycin binds to the 60S ribosomal subunit. B. Puromycin inactivates elongation factor-2. C. Streptomycin binds to the 30S ribosomal subunit. D. Tetracyclines inhibit peptidyltransferase. Correct answer = C. Streptomycin binds the 30S subunit and inhibits translation initiation. Erythromycin binds the 50S ribosomal subunit (60S denotes a eukaryote) and blocks the tunnel through which the peptide leaves the ribosome. Puromycin has structural similarity to aminoacyl-transfer RNA. It is incorporated into the growing chain, inhibits elongation, and results in premature termination in both prokaryotes and eukaryotes. Tetracyclines bind the 30S ribosomal subunit and block access to the A site, inhibiting elongation.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2.6. Translation of a synthetic polyribonucleotide containing the repeating sequence CAA in a cell-free protein-synthesizing system produces three homopolypeptides: polyglutamine, polyasparagine, and polythreonine. If the codons for glutamine and asparagine are CAA and AAC, respectively, which of the following triplets is the codon for threonine? A. AAC B. ACA C. CAA D. CAC E. CCA Correct answer = B. The synthetic polynucleotide sequence of CAACAACAACAA … could be read by the in vitro protein-synthesizing system starting at the first C, the first A, or the second A (that is, in any one of three reading frames). In the first case, the first triplet codon would be CAA, which codes glutamine; in the second case, the first triplet codon would be AAC, which codes for asparagine; in the last case, the first triplet codon would be ACA, which codes for threonine. 2.7. Which of the following is required for both prokaryotic and eukaryotic protein synthesis?


Biochemistry_Lippinco

Biochemistry_Lippinco. 2.7. Which of the following is required for both prokaryotic and eukaryotic protein synthesis? A. Binding of the small ribosomal subunit to the Shine-Dalgarno sequence B. Formylated methionyl-transfer (t)RNA C. Movement of the messenger RNA out of the nucleus and into the cytoplasm D. Recognition of the 5′-cap by initiation factors E. Translocation of the peptidyl-tRNA from the A site to the P site Correct answer = E. In both prokaryotes and eukaryotes, continued translation (elongation) requires movement of the peptidyl-tRNA from the A to the P site to allow the next aminoacyl-tRNA to enter the A site. Only prokaryotes have a Shine-Dalgarno sequence and use formylated methionine and only eukaryotes have a nucleus and co-and posttranscriptionally process their mRNA.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2.8. α1-Antitrypsin (AAT) deficiency can result in emphysema, a lung pathology, because the action of elastase, a serine protease, is unopposed. Deficiency of AAT in the lungs is the consequence of impaired secretion from the liver, the site of its synthesis. Proteins such as AAT that are destined to be secreted are best characterized by which of the following statements? A. Their synthesis is initiated on the smooth endoplasmic reticulum. B. They contain a mannose 6-phosphate targeting signal. C. They always contain methionine as the N-terminal amino acid. D. They are produced from translation products that have an N-terminal hydrophobic signal sequence. E. They contain no sugars with O-glycosidic linkages because their synthesis does not involve the Golgi.


Biochemistry_Lippinco

Biochemistry_Lippinco. E. They contain no sugars with O-glycosidic linkages because their synthesis does not involve the Golgi. Correct answer = D. Synthesis of secreted proteins is begun on free (cytosolic) ribosomes. As the N-terminal signal sequence of the peptide emerges from the ribosome, it is bound by the signal recognition particle, taken to the rough endoplasmic reticulum (RER), threaded into the lumen, and cleaved as translation continues. The proteins move through the RER and the Golgi and undergo processing such as N-glycosylation (RER) and O-glycosylation (Golgi). In the Golgi, they are packaged in secretory vesicles and released from the cell. The smooth endoplasmic reticulum is associated with synthesis of lipids, not proteins, and has no ribosomes attached. Phosphorylation at carbon 6 of terminal mannose residues in glycoproteins targets these proteins (acid hydrolases) to lysosomes. The N-terminal methionine is removed from most proteins during processing.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2.9. Why is the genetic code described as both degenerate and unambiguous? A given amino acid can be coded for by more than one codon (degenerate code), but a given codon codes for just one particular amino acid (unambiguous code). Regulation of Gene Expression 33 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. Regulation of Gene Expression 33 For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW Gene expression refers to the multistep process that ultimately results in the production of a functional gene product, either ribonucleic acid (RNA) or protein. The first step in gene expression, the use of deoxyribonucleic acid (DNA) for the synthesis of RNA (transcription), is the primary site of regulation in both prokaryotes and eukaryotes. In eukaryotes, however, gene expression also involves extensive posttranscriptional and posttranslational processes as well as actions that influence access to particular regions of the DNA. Each of these steps can be regulated to provide additional control over the kinds and amounts of functional products that are produced.


Biochemistry_Lippinco

Biochemistry_Lippinco. Not all genes are tightly regulated. For example, genes described as constitutive encode products required for basic cellular functions and so are expressed at essentially a constant level. They are also known as “housekeeping” genes. Regulated genes, however, are expressed only under certain conditions. They may be expressed in all cells or in only a subset of cells, for example, hepatocytes. The ability to regulate gene expression (that is, to determine if, how much, and when particular gene products will be made) gives the cell control over structure and function. It is the basis for cellular differentiation, morphogenesis, and adaptability of any organism. Control of gene expression is best understood in prokaryotes, but many themes are repeated in eukaryotes. Figure 33.1 shows some of the sites where gene expression can be controlled. II. REGULATORY SEQUENCES AND MOLECULES


Biochemistry_Lippinco

Biochemistry_Lippinco. Regulation of transcription, the initial step in all gene expression, is controlled by regulatory sequences of DNA that are usually embedded in the noncoding regions of the genome. The interaction between these DNA sequences and regulatory molecules, such as transcription factors, can induce or repress the transcriptional machinery, influencing the kinds and amounts of products that are produced. The regulatory DNA sequences are called cis-acting because they influence expression of genes on the same chromosome as the regulatory sequence (see p. 439). The regulatory molecules are called trans-acting because they can diffuse (transit) through the cell from their site of synthesis to their DNA-binding sites (Fig. 33.2). For example, a protein transcription factor (a trans-acting molecule) that regulates a gene on chromosome 6 might itself have been produced from a gene on chromosome 11. The binding of proteins to DNA is through structural motifs such as the zinc finger (Fig. 33.3),


Biochemistry_Lippinco

Biochemistry_Lippinco. that regulates a gene on chromosome 6 might itself have been produced from a gene on chromosome 11. The binding of proteins to DNA is through structural motifs such as the zinc finger (Fig. 33.3), leucine zipper, or helix-turn-helix in the protein.


Biochemistry_Lippinco

Biochemistry_Lippinco. III. REGULATION OF PROKARYOTIC GENE EXPRESSION In prokaryotes such as the bacterium Escherichia coli (E. coli), regulation of gene expression occurs primarily at the level of transcription and, in general, is mediated by the binding of trans-acting proteins to cis-acting regulatory elements on their single DNA molecule (chromosome). [Note: Regulating the first step in the expression of a gene is an efficient approach, insofar as energy is not wasted making unneeded gene products.] Transcriptional control in prokaryotes can involve the initiation or premature termination of transcription. A. Messenger RNA transcription from bacterial operons


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Messenger RNA transcription from bacterial operons In bacteria, the structural genes that encode proteins involved in a particular metabolic pathway are often found sequentially grouped on the chromosome along with the cis-acting elements that regulate the transcription of these genes. The transcription product is a single polycistronic messenger RNA ([mRNA] see p. 434). The genes are, thus, coordinately regulated (that is, turned on or off as a unit). This entire package is referred to as an operon. B. Operators in bacterial operons


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Operators in bacterial operons Bacterial operons contain an operator, a segment of DNA that regulates the activity of the structural genes of the operon by reversibly binding a protein known as the repressor. If the operator is not bound by the repressor, RNA polymerase (RNA pol) binds the promoter, passes over the operator, and reaches the protein-coding genes that it transcribes to mRNA. If the repressor is bound to the operator, the polymerase is blocked and does not produce mRNA. As long as the repressor is bound to the operator, no mRNA (and, therefore, no proteins) are made. However, when an inducer molecule is present, it binds to the repressor, causing the repressor to change shape so that it no longer binds the operator. When this happens, RNA pol can initiate transcription. One of the best-understood examples is the inducible lactose (lac) operon of E. coli that illustrates both positive and negative regulation (Fig. 33.4). C. Lactose operon


Biochemistry_Lippinco

Biochemistry_Lippinco. The lac operon contains the genes that code for three proteins involved in the catabolism of the disaccharide lactose: the lacZ gene codes for βgalactosidase, which hydrolyzes lactose to galactose and glucose; the lacY gene codes for a permease, which facilitates the movement of lactose into the cell; and the lacA gene codes for thiogalactoside transacetylase, which acetylates lactose. [Note: The physiologic function of this acetylation is unknown.] All of these proteins are maximally produced only when lactose is available to the cell and glucose is not. [Note: Bacteria use glucose, if available, as a fuel in preference to any other sugar.] The regulatory portion of the operon is upstream of the three structural genes and consists of the promoter region where RNA pol binds and two additional sites, the operator (O) and the catabolite activator protein (CAP) sites, where regulatory proteins bind. The lacZ, lacY, and lacA genes are maximally expressed only when the O site is empty and


Biochemistry_Lippinco

Biochemistry_Lippinco. sites, the operator (O) and the catabolite activator protein (CAP) sites, where regulatory proteins bind. The lacZ, lacY, and lacA genes are maximally expressed only when the O site is empty and the CAP site is bound by a complex of cyclic adenosine monophosphate ([cAMP] see p. 94) and the CAP, sometimes called the cAMP regulatory protein (CRP). A regulatory gene, the lacI gene, codes for the repressor protein (a trans-acting factor) that binds to the O site with high affinity. [Note: The lacI gene has its own promoter and is not part of the lac operon.] 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. When only glucose is available: In this case, the lac operon is repressed (turned off). Repression is mediated by the repressor protein binding via a helix-turn-helix motif (Fig. 33.5) to the O site, which is downstream of the promoter (see Fig. 33.4A). Binding of the repressor interferes with the binding of RNA pol to the promoter, thereby inhibiting transcription of the structural genes. This is an example of negative regulation. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. When only lactose is available: In this case, the lac operon is induced (maximally expressed, or turned on). A small amount of lactose is converted to an isomer, allolactose. This compound is an inducer that binds to the repressor protein, changing its conformation so that it can no longer bind to the O site. In the absence of glucose, adenylyl cyclase is active, and cAMP is made and binds to the CAP. The cAMP–CAP transacting complex binds to the CAP site, causing RNA pol to initiate transcription with high efficiency at the promoter site (see Fig. 33.4B). This is an example of positive regulation. The transcript is a single polycistronic mRNA molecule that contains three sets of start and stop codons. Translation of the mRNA produces the three proteins that allow lactose to be used for energy production by the cell. [Note: In contrast to the inducible lacZ, lacY, and lacA genes, whose expression is regulated, the lacI gene is constitutive. Its gene product, the repressor protein, is


Biochemistry_Lippinco

Biochemistry_Lippinco. production by the cell. [Note: In contrast to the inducible lacZ, lacY, and lacA genes, whose expression is regulated, the lacI gene is constitutive. Its gene product, the repressor protein, is always made and is active unless the inducer is present.] 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. When both glucose and lactose are available: In this case, the lac operon is uninduced, and transcription is negligible, even if lactose is present at a high concentration. Adenylyl cyclase is inhibited in the presence of glucose (a process known as catabolite repression) so no cAMP–CAP complex forms, and the CAP site remains empty. Therefore, the RNA pol is unable to effectively initiate transcription, even though the repressor is not bound to the O site. Consequently, the three structural genes of the operon are expressed only at a very low (basal) level (see Fig. 33.4C). [Note: Induction causes a 50-fold enhancement over basal expression.] D. Tryptophan operon


Biochemistry_Lippinco

Biochemistry_Lippinco. The tryptophan (trp) operon contains five structural genes that code for enzymes required for the synthesis of the amino acid tryptophan. As with the lac operon, the trp operon is subject to negative control. However, for the repressible trp operon, negative control includes Trp itself binding to a repressor protein and facilitating the binding of the repressor to the operator: Trp is a corepressor. Because repression by Trp is not always complete, the trp operon, unlike the lac operon, is also regulated by a process known as attenuation. With attenuation, transcription is initiated but is terminated well before completion (Fig. 33.6). If Trp is plentiful, transcription initiation that escaped repression by Trp is attenuated (stopped) by the formation of an attenuator, a hairpin (stem-loop) structure in the mRNA similar to that seen in rho-independent termination (see p. 437). [Note: Because transcription and translation are temporally linked in prokaryotes (see p. 454), attenuation


Biochemistry_Lippinco

Biochemistry_Lippinco. structure in the mRNA similar to that seen in rho-independent termination (see p. 437). [Note: Because transcription and translation are temporally linked in prokaryotes (see p. 454), attenuation also results in the formation of a truncated, nonfunctional peptide product that is rapidly degraded.] If Trp becomes scarce, the operon is expressed. The 5′-end of the mRNA contains two adjacent codons for Trp. The lack of Trp causes ribosomes to stall at these codons, covering regions of the mRNA required for formation of the attenuation hairpin. This prevents attenuation and allows transcription to continue.


Biochemistry_Lippinco

Biochemistry_Lippinco. Transcriptional attenuation can occur in prokaryotes because translation of an mRNA begins before its synthesis is complete. This does not occur in eukaryotes because the presence of a membrane-bound nucleus spatially and temporally separates transcription and translation. E. Coordination of transcription and translation Although transcriptional regulation of mRNA production is primary in bacteria, regulation of ribosomal RNA (rRNA) and protein synthesis plays important roles in adaptation to environmental stress. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Stringent response: E. coli has seven operons that synthesize the rRNA needed for ribosome assembly, and each is regulated in response to changes in environmental conditions. Regulation in response to amino acid starvation is known as the stringent response. The binding of an uncharged transfer RNA (tRNA) to the A site of a ribosome (see p. 452) triggers a series of events that leads to the production of the alarmone guanosine tetraphosphate (ppGpp). The synthesis of this unusual derivative of guanosine diphosphate (GDP) is catalyzed by stringent factor (RelA), an enzyme physically associated with ribosomes. Elevated levels of ppGpp result in inhibition of rRNA synthesis (Fig. 33.7). [Note: In addition to rRNA synthesis, tRNA synthesis and some mRNA synthesis (for example, for ribosomal proteins) are also inhibited. However, synthesis of mRNA for enzymes required for amino acid biosynthesis is not inhibited. ppGpp binds RNA pol and alters promoter 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Regulatory ribosomal proteins: Operons for ribosomal proteins (rproteins) can be inhibited by an excess of their own protein products. For each operon, one specific r-protein functions in the repression of selection through use of different sigma factors for the polymerase (see p. 435).] translation of the polycistronic mRNA from that operon (Fig. 33.8). The r-protein does so by binding to the Shine-Dalgarno (SD) sequence located on the mRNA just upstream of the first initiating AUG codon (see p. 448) and acting as a physical impediment to the binding of the small ribosomal subunit to the SD sequence. Thus, one r-protein inhibits synthesis of all the r-proteins of the operon. This same r-protein also binds to rRNA and with a higher affinity than for mRNA. If the concentration of rRNA falls, the r-protein then is available to bind its own mRNA and inhibit its translation. This coordinated regulation keeps the synthesis of r-proteins in balance with the transcription of rRNA, so that


Biochemistry_Lippinco

Biochemistry_Lippinco. the r-protein then is available to bind its own mRNA and inhibit its translation. This coordinated regulation keeps the synthesis of r-proteins in balance with the transcription of rRNA, so that each is present in appropriate amounts for the formation of ribosomes.


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. REGULATION OF EUKARYOTIC GENE EXPRESSION The higher degree of complexity of eukaryotic genomes, as well as the presence of a nuclear membrane, necessitates a wider range of regulatory processes. As with the prokaryotes, transcription is the primary site of regulation. Again, the theme of trans-acting factors binding to cis-acting elements is seen. Operons, however, are not found in eukaryotes, which must use alternate strategies to solve the problem of how to coordinately regulate all the genes required for a specific response. In eukaryotes, gene expression is also regulated at multiple levels other than transcription. For example, the major modes of posttranscriptional regulation at the mRNA level are alternative mRNA splicing and polyadenylation, control of mRNA stability, and control of translational efficiency. Additional regulation at the protein level occurs by mechanisms that modulate stability, processing, or targeting of the protein. A. Coordinate regulation


Biochemistry_Lippinco

Biochemistry_Lippinco. The need to coordinately regulate a group of genes to cause a particular response is of key importance in organisms with more than one chromosome. An underlying theme occurs repeatedly: A trans-acting protein functions as a specific transcription factor (STF) that binds to a cisacting regulatory consensus sequence (see p. 415) on each of the genes in the group even if they are on different chromosomes. [Note: The STF has a DNA-binding domain (DBD) and a transcription activation domain (TAD). The TAD recruits coactivators, such as histone acetyltransferases (see p. 438), and the general transcription factors (see p. 439) that, along with RNA pol, are required for formation of the transcription initiation complex at the promoter. Although the TAD recruits a variety of proteins, the specific effect of any one of them is dependent upon the protein composition of the complex. This is known as combinatorial control.] Examples of coordinate regulation in eukaryotes include the galactose


Biochemistry_Lippinco

Biochemistry_Lippinco. effect of any one of them is dependent upon the protein composition of the complex. This is known as combinatorial control.] Examples of coordinate regulation in eukaryotes include the galactose circuit and the hormone response system.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Galactose circuit: This regulatory scheme allows for the use of galactose when glucose is not available. In yeast, a unicellular organism, the genes required to metabolize galactose are on different chromosomes. Coordinated expression is mediated by the protein Gal4 (Gal = galactose), a STF that binds to a short regulatory DNA sequence upstream of each of the genes. The sequence is called the upstream activating sequence Gal (UASGal). Binding of Gal4 to UASGal through zinc fingers in its DBD occurs in both the absence and presence of galactose. When the sugar is absent, the regulatory protein Gal80 binds Gal4 at its TAD, thereby inhibiting gene transcription (Fig. 33.9A). When present, galactose activates the Gal3 protein. Gal3 binds Gal80, thereby allowing Gal4 to activate transcription (Fig. 33.9B). [Note: Glucose prevents the use of galactose by inhibting expression of Gal4 protein.]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. presence of galactose. [Note: Target genes, whether on the same or a different chromosome, each have an upstream activating sequence galactose (UASGal).] TAD = transcription activation domain; DBD = DNA-binding domain; mRNA = messenger RNA. 2. Hormone response system: Hormone response elements (HRE) are DNA sequences that bind trans-acting proteins and regulate gene expression in response to hormonal signals in multicellular organisms. Hormones bind to either intracellular (nuclear) receptors (for example, steroid hormones; see p. 240) or cell-surface receptors (for example, the peptide hormone glucagon; see p. 314). a.


Biochemistry_Lippinco

Biochemistry_Lippinco. Intracellular receptors: Members of the nuclear receptor superfamily, which includes the steroid hormone (glucocorticoids, mineralocorticoids, androgens, and estrogens), vitamin D, retinoic acid, and thyroid hormone receptors, function as STF. In addition to domains for DNA-binding and transcriptional activation, these receptors also contain a ligand-binding domain. For example, the steroid hormone cortisol (a glucocorticoid) binds intracellular receptors at the ligand-binding domain (Fig. 33.10). Binding causes a conformational change in the receptor that activates it. The receptor– hormone complex enters the nucleus, dimerizes, and binds via a zinc finger motif to DNA at a regulatory element, the glucocorticoid response element (GRE) that is an example of a HRE. Binding allows recruitment of coactivators to the TAD and results in expression of cortisol-responsive genes, each of which is under the control of its own GRE. Binding of the receptor–hormone complex to the GRE allows


Biochemistry_Lippinco

Biochemistry_Lippinco. of coactivators to the TAD and results in expression of cortisol-responsive genes, each of which is under the control of its own GRE. Binding of the receptor–hormone complex to the GRE allows coordinate expression of a group of target genes, even though these genes are on different chromosomes. The GRE can be located upstream or downstream of the genes it regulates and at great distances from them. The GRE, then, can function as a true enhancer (see p. 440). [Note: If associated with repressors, hormone–receptor complexes inhibit transcription.] b.