Genetics - Complete from Lippincott Ed 8 PDF

UNIT VII: storage and Expression of Genetic Information DNA Structure, Replication, and 30 Repair Il. OVERVIEW ea ; | Nucleic acids are required for the storage and expression of genetic infor- mation. There are two chemically distinct types of nucleic acids: deoxy- ribonucleic acid (DNA) an...

UNIT VII: storage and Expression of Genetic Information DNA Structure, Replication, and 30 Repair Il. OVERVIEW ea ; | Nucleic acids are required for the storage and expression of genetic infor- mation. There are two chemically distinct types of nucleic acids: deoxy- ribonucleic acid (DNA) and ribonucleic acid ([RNA] see Chapter 31). DNA, the repository of genetic information (or, genome), is present not only in chromosomes in the nucleus of eukaryotic organisms, but also in mitochondria and the chloroplasts of plants. Prokaryotic cells, which lack nuclei, have a single chromosome but may also contain nonchromosomal DNA in the form of plasmids. The genetic information found in DNA is copied and transmitted to daughter cells through DNA replication. The Transcription DNA contained in a fertilized egg encodes the information that directs the development of an organism. This development may involve the produc- tion of billions of cells. Each cell is specialized, expressing only those func- tions that are required for it to perform its role in maintaining the organism. RNA Therefore, DNA must be able not only to replicate precisely each time a cell divides, but also to have the information that it contains be selectively expressed. Transcription (RNA synthesis) is the first stage in the expres- Translation sion of genetic information (see Chapter 31). Next, the code contained in the nucleotide sequence of messenger RNA molecules is translated (protein synthesis; see Chapter 32), thus completing gene expression. The regulation of gene expression is discussed in Chapter 33. Replication ~DNA PROTEIN The flow of information from DNA to RNA to protein is termed the “central dogma’ of molecular biology (Fig. 30.1) and is descriptive of all organisms, with the exception of some viruses Figure 30.1 that have RNA as the repository of their genetic information. The “central dogma’ of molecular biology. 411 412 30. DNA Structure, Replication, and Repair A rE 2 4 BI 5-End XN ii Thymine (T) N p fe} Lt p rZa—A p LZ—s p 3 exonuclease activity. This enzyme fills the gaps with DNA, proofreading as it synthesizes. The final phosphodiester linkage is catalyzed by DNA ligase. There are at least five high-fidelity eukaryotic DNA pols. Pol a is a multisubunit enzyme, one subunit of which is a primase. Pol a 5'>3' polymerase activity adds a short piece of DNA to the RNA primer. Pol e completes DNA synthesis on the leading strand, whereas pol 6 elongates each lagging strand fragment. Pol B is involved with DNA repair, and pol y replicates mitochondrial DNA. Pols e, 6, and y use 3’--5' exonuclease activity to proofread. Nucleoside analogs containing modified sugars can be used to block DNA chain growth. They are useful in anticancer and antiviral chemotherapy. Telomeres are stretches of highly repetitive DNA complexed with protein that protect the ends of linear chromosomes. As most cells divide and age, these sequences are shortened, contributing to senescence. In cells that do not senesce (for example, germline and cancer cells), the ribonucleoprotein telomerase employs its protein component reverse transcriptase to extend the telomeres, using its RNA component as a template. There are five classes of positively charged histone (H) proteins. Two of each of histones H2A, H2B, H3, and H4 form an octameric structural core around which DNA is wrapped, creating a nucleosome. The DNA connecting the nucleosomes, called linker DNA, is bound to H1. Nucleosomes can be packed more tightly to form a nucleofilament. Additional levels of organization create a chromosome. Most DNA damage can be corrected by excision repair involving recognition and removal of the damage by repair proteins, followed by replacement by DNA pols and joining by /igase. Ultraviolet radiation can cause thymine dimers that are recognized and removed in E. coli by uvrABC proteins of nucleotide excision repair. Defects in the XP proteins needed for nucleotide excision repair of thymine dimers in humans result in xeroderma pigmentosum. Mismatched bases are repaired by a similar process of recognition and removal by Mut proteins in E. coli. The extent of methylation is used for strand identification in prokaryotes. Defective mismatch repair by homologous proteins in humans is associated with hereditary nonpolyposis colorectal cancer. Abnormal bases (such as uracil) are removed by DNA N-glycosylases in base exclslon repalr, and the sugar phosphate at the apyrimidinic or apurinic site is cut out. Double-strand breaks in DNA are repaired by nonhomologous end joining (error prone) and template-requiring homologous recombination (“error-free”). J \ 432 30. DNA Structure, Replication, and Repair Study Questions Choose the ONE best answer. 30.1 30.2 30.3 30.4 30.5 A 10-year-old girl is brought by her parents to the dermatologist. She has many freckles on her face, neck, arms, and hands, and the parents report that she is unusually sensitive to sunlight. Two basal cell carcinomas are identified on her face. Based on the clinical picture, which of the following processes is most likely to be defective in this patient? A. Repair of double-strand breaks by error-prone homol- ogous recombination B. Removal of mismatched bases from the 3’-end of Okazaki fragments by a methyl-directed process C. Removal of pyrimidine dimers from DNA by nucleo- tide excision repair D. Removal of uracil from DNA by base excision repair Telomeres are complexes of DNA and protein that protect the ends of linear chromosomes. In most normal human somatic cells, telomeres shorten with each division. In stem cells and in cancer cells, however, telomeric length is maintained. In the synthesis of telomeres: A. telomerase, a ribonucleoprotein, provides both the RNA and the protein needed for synthesis. B. the RNA of telomerase serves as a primer. C. the RNA of telomerase is a ribozyme. D. the protein of telomerase is a DNA-directed DNA polymerase. E. the shorter 3’—5’ strand gets extended. F._ the direction of synthesis is 3’35’. While studying the structure of a small gene that was sequenced during the Human Genome Project, an investigator notices that one strand of the DNA molecule contains 20 A, 25 G, 30 C, and 22 T. How many of each base is found in the complete double-stranded molecule? A= 40, G=50, C =60,T=44 A= 42, G=55,C=55,T=42 A= 44, G=60, C=50,T=40 A=45, G=45, 0 =52,T=52 A=50, G=47,C =50,T=47 moOW> List the order in which the following enzymes participate in prokaryotic replication. Ligase Polymerase | (3’5’ exonuclease activity) Polymerase | (5’-3’ exonuclease activity) Polymerase | (5'-3’ polymerase activity) Polymerase III Primase ™mO50p> Dideoxynucleotides lack a 3/-hydroxyl group. Why would incorporation of a dideoxynucleotide into DNA stop replication? Correct answer = C. The sensitivity to sunlight, extensive freckling on parts of the body exposed to the sun, and presence of skin cancer at a young age indicate that the patient most likely suffers from xeroderma pigmentosum (XP). These patients are deficient in any one of several XP proteins required for nucleotide excision repair of pyrimidine dimers in ultraviolet radiation—damaged DNA. Double-strand breaks are repaired by nonhomologous end joining (error prone) or homologous recombination (‘error free”). Methylation is not used for strand discrimina- tion in eukaryotic mismatch repair. Uracil is removed from DNA molecules by a specific glycosylase in base excision repair, but a defect in this process does not cause XP. Correct answer = A. Telomerase is a ribonucleoprotein particle required for telomere maintenance. Telomerase contains an RNA that serves as the template, not the primer, for the synthesis of telomeric DNA by the reverse transcriptase of telomerase. Telomeric RNA has no catalytic activity. As a reverse transcriptase, telomerase synthesizes DNA using its RNA template and so is an RNA-directed DNA polymerase. The direction of synthe- sis, as with all DNA synthesis, is 5’>3’, and itis the 3’-end of the already longer 5’—3’ strand that gets extended. Correct answer = B. The two DNA strands are comple- mentary to each other, with A base-paired with T and G base-paired with C. So, for example, the 20 A on the first strand would be paired with 20 T on the second strand, the 25 G on the first strand would be paired with 25 C on the second strand, and so forth. When these are all added together, the correct numbers of each base are indicated in choice B. Notice that, in the correct answer, A = T and G=C. Correct answer: F, E, C, D, B, A. Primase makes the RNA primer; polymerase (pol) III extends the primer with DNA (and proofreads); pol | removes the primer with its 5’-3’ exonuclease activity, fills in the gap with its 5’>3’ polymerase activity, and removes errors with its 3’-5’ exonuclease activity; and ligase makes the 5’>3’-phos- phodiester bond that links the DNA made by pols | and Ill. The lack of the 3’-OH group prevents formation of the 3’-hydroxyl > 5’-phosphate bond that links one nucleo- tide to the next in DNA. RNA Structure, Synthesis, and Processing |. OVERVIEW The genetic master plan of an organism is contained in the sequence of deoxyribonucleotides in its DNA. However, it is through ribonucleic acid (RNA), the “working copies” of DNA, that the master plan is expressed (Fig. 31.1). The copying process, during which a DNA strand serves as a template for the synthesis of RNA, is called transcription. Transcription pro- duces messenger RNA (mRNA), which are translated into sequences of amino acids (proteins), and ribosomal RNA (rRNA), transfer RNA (tRNA), and additional RNA molecules that perform specialized structural, catalytic, and regulatory functions and are not translated. That is, they are noncod- ing RNA (ncRNA). Therefore, the final product of gene expression can be RNA or protein, depending upon the gene. [Note: Only ~2% of the genome encodes proteins.] A central feature of transcription is that it is highly selec- tive. For example, many transcripts are made of some regions of the DNA. In other regions, few or no transcripts are made. This selectivity is due, at least in part, to signals embedded in the nucleotide sequence of the DNA. These signals instruct the ANA polymerase where to start, how often to start, and where to stop transcription. Several regulatory proteins are also involved in this selection process. The biochemical differentiation of an organism's tissues is ultimately a result of the selectivity of the transcription process. [Note: This selectivity of transcription is in contrast to the “all-or-none” nature of genomic replication.] Another important feature of transcription is that many RNA transcripts that initially are faithful copies of one of the two DNA strands may undergo various modifications, such as terminal additions, base modifications, trimming, and internal segment removal, which convert the inactive primary transcript into a functional molecule. The transcriptome is the complete set of RNA transcripts expressed by a genome. ll. RNA STRUCTURE There are three major types of RNA that participate in the process of protein synthesis: rRNA, tRNA, and mRNA. Like DNA, these RNA are unbranched polymeric molecules composed of nucleoside monophos- phates joined together by 3’-5’-phosphodiester bonds (see p. 412). However, they differ from DNA in several ways. For example, they are considerably smaller than DNA, contain ribose instead of deoxyribose 37 130000011000000000000K TRANSCRIPTION ¥ m’Gppp wrrwn~pApApA mRNA Figure 31.1 Expression of genetic information by transcription. [Note: RNA shown are eukaryotic.] tRNA = transfer RNA; rRNA = ribosomal RNA; mRNA = messenger RNA; m’Gppp = 7-methylguanosine- triphosphate cap; pApApA = poly-A tail; p = phosphate. 433 434 31. RNA Siructure, Synthesis, and Processing Prokaryotic rRNA AAR 23S Annan 16S AAA 5S Eukaryotic rRNA DIARRA IANA 28S AANA 18S porn 5.8S anna 5S Figure 31.2 Prokaryotic and eukaryotic ribosomal RNA (rRNA). S = Svedberg unit. -End CCA Site of amino “End | | acid attachment Complementary 5 base palrs ih (Intrachalin) " TYC loop —— D loop Anticodon f loop Anticodon Figure 31.3 A. Characteristic transfer RNA (tRNA) secondary structure (cloverleaf). B. Folded (tertiary) tRNA structure found in cells. D = dihydrouracil; = pseudouracil; T = thymine; C = cytosine; A = adenine. and uracil instead of thymine, and exist as single strands that are capable of folding into complex structures. The three major types of RNA also differ from each other in size, function, and special structural modifica- tions. [Note: In eukaryotes, additional small ncRNA molecules found in the nucleolus (snoRNA), nucleus (snRNA), and cytoplasm (microRNA [miRNA]) perform specialized functions as described on pp. 441, 442, and 475.] A. Ribosomal RNA rRNA are found in association with several proteins as components of the ribosomes, the complex structures that serve as the sites for protein synthesis (see p. 451). Prokaryotic cells contain three distinct size species of rRNA (23S, 16S, and 5S, where S is the Svedberg unit for sedimentation rate that is determined by the size and shape of the particle), as shown in Figure 31.2. Eukaryotic cells contain four rRNA species (28S, 18S, 5.8S, and 5S). Together, rRNA make up ~80% of the total RNA in the cell. [Note: Some RNA function as catalysts, for example, an rRNA in protein synthesis (see p. 455). RNA with cata- lytic activity is termed a ribozyme.] B. Transfer RNA tRNA are the smallest (4S) of the three major types of RNA mole- cules. There is at least one specific type of tRNA molecule for each of the 20 amino acids commonly found in proteins. Together, tRNA make up ~15% of the total RNA in the cell. The tRNA molecules contain a high percentage of unusual (modified) bases, for example, dihydro- uracil (see Fig. 22.2, p. 292), and have extensive intrachain base- pairing (Fig. 31.3) that leads to characteristic secondary and tertiary structure. Each tRNA serves as an adaptor molecule that carries its specific amino acid, covalently attached to its 3’-end, to the site of protein synthesis. There, it recognizes the genetic code sequence on an mRNA, which specifies the addition of that amino acid to the grow- ing peptide chain (see p. 447). C. Messenger RNA mRNA comprises only ~5% of the RNA in a cell, yet is by far the most heterogeneous type of RNA in size and base sequence. mRNA is coding RNA in that it carries genetic information from DNA for use in protein synthesis. In eukaryotes, this involves transport of MRNA out of the nucleus and into the cytosol. An mRNA carrying infor- mation from more than one gene is polycistronic (cistron = gene). Polycistronic mRNA is characteristic of prokaryotes. An MRNA car- rying information from only one gene is monocistronic and is charac- teristic of eukaryotes. In addition to the protein-coding regions that can be translated, mRNA contains untranslated regions at its 5’- and 3’-ends (Fig. 31.4). Special structural characteristics of eukaryotic (but not prokaryotic) mRNA include a long sequence of adenine nucleotides (a poly-A tail) on the 3’-end of the RNA, plus a cap on the 5’-end consisting of a molecule of 7-methylguanosine attached through an unusual (5’-5’) triphosphate linkage. The mechanisms for modifying MRNA to create these special structural characteristics are discussed on pp. 441-442. Ill. Prokaryotic Gene Transcription 435 lll. PROKARYOTIC GENE TRANSCRIPTION 3-Untranslated region The structure of magnesium-requiring RNA polymerase (RNA pol), the “region signals that control transcription, and the varieties of modification that RNA transcripts can undergo differ among organisms, particularly from | Coding i i i Cc I Poly-A tall prokaryotes to cukaryotes. Therefore, te discussions of prokaryotic and be ea tes ll EY eukaryotic transcription are presented separately. Gppp—~~-\47.-.. pApApApApA-OH A. Prokaryotic RNA polymerase 5-End 3’-End In bacteria, one species of RNA pol synthesizes all of the RNA except for the short RNA primers needed for DNA replication [Note: RNA 7 : ‘ nan ‘. Figure 31.4 primers are synthesized by the specialized, monomeric enzyme pri- : : : ;. Structure of eukaryotic messenger mase (see p. 418).] ANA polis a multisubunit enzyme that recognizes RNA. G = guanine; A = adenine. a nucleotide sequence (the promoter region) at the beginning of a , length of DNA that is to be transcribed. It next makes a complemen- tary RNA copy of the DNA template strand and then recognizes the end of the DNA sequence to be transcribed (the termination region). ee Base palrs phosphate RNA is synthesized from its 5’-end to its 3’-end, antiparallel to its backbone backbone DNA template strand (see p. 415). The template is copied as it is in DNA synthesis, in which a guanine (G) on the DNA specifies a cyto- sine (C) in the RNA, a C specifies a G, a thymine (T) specifies an adenine (A), but an A specifies a uracil (U) instead of a T (Fig. 31.5). The RNA, then, is complementary to the DNA template (antisense, minus) strand and identical to the coding (sense, plus) strand, with U replacing T. Within the DNA molecule, regions of both strands can serve as templates for transcription. For a given gene, however, only one of the two DNA strands can be the template. Which strand is used is determined by the location of the promoter for that gene. Transcription by RANA pol involves a core enzyme and several aux- iliary proteins. 1. Core enzyme: Five of the enzyme’s peptide subunits, 2 a, 1 p, 1 p’, and 1 Q, are required for enzyme assembly (a, Q), template binding (6’), and the 5’>3’ polymerase activity (6) and together are referred to as the core enzyme (Fig. 31.6). However, this enzyme lacks specificity (that is, it cannot recognize the promoter region on the DNA template). 2. Holoenzyme: The o subunit (sigma factor) enables ANA pol to recognize promoter regions on the DNA. The o subunit plus the core enzyme make up the holoenzyme. [Note: Different o factors recognize different groups of genes, with o”° predominating. ] B. Steps in RNA synthesis The process of transcription of a typical gene of Escherichia coli (E. coli) can be divided into three phases: initiation, elongation, and termination. A transcription unit extends from the promoter to the ter- mination region, and the initial product of transcription by ANA pol is termed the primary transcript. 1. Initiation: Transcription begins with the binding of the ANA pol holoenzyme to a region of the DNA known as the promoter, which is not transcribed. The prokaryotic promoter contains characteristic consensus sequences (Fig. 31.7). [Note: Consensus sequences 5 hicrte ft 3 oy (G@] iit: Hf eles a” u Hydrogen oe Gee DNA RNA 5 Figure 31.5 Antiparallel, complementary base pairs between DNA and RNA. T = thymine; A = adenine; C = cytosine; G = guanine; U = uracil. Holoenzyme Figure 31.6 Components of prokaryotic RNA polymerase. 436 31. RNA Siructure, Synthesis, and Processing ——_ Sequences within the prokaryotic promoter region that are recognize: d } — ———.._ bythe ANA polymerase holoenzyme = =-—__~—___. Start of transcription -35 C Pribnow 4 Sequence ~19 base pairs box ~7 base pairs Y TTGACA TATAAT Y ee DNA Thrirn ry x es fk tee | Lil 5 —40 =35 -30 —25 —20 =15 =10 —5 =H +5 Figure 31.7 Structure of the prokaryotic promoter region. T = thymine; G = guanine; A = adenine; C = cytosine. are idealized sequences in which the base shown at each position is the base most frequently (but not necessarily always) encoun- tered at that position.] Those that are recognized by prokaryotic ANA pol c factors include the following. a. -35 Sequence: A consensus sequence (5’-TTGACA-3’), cen- tered about 35 bases to the left of the transcription start site (see Fig. 31.7), is the initial point of contact for the holoenzyme, and a closed complex is formed. [Note: By convention, the regu- latory sequences that control transcription are designated by the 5’-3’ nucleotide sequence on the coding strand. A base in the promoter region is assigned a negative number if it occurs prior to (to the left of, toward the 5’-end of, or “upstream” of) the transcription start site. Therefore, the TTGACA sequence is centered at approximately base —35. The first base at the transcription start site is assigned a position of +1. There is no base designated “O”.] b. Pribnow box: The holoenzyme moves and covers a second consensus sequence (5’-TATAAT-3’), centered at about -10 (see Fig. 31.7), which is the site of melting (unwinding) of a short stretch (~14 base pairs) of DNA. This initial melting con- verts the closed initiation complex to an open complex known as a transcription bubble. [Note: A mutation in either the —10 or the -35 sequence can affect the transcription of the gene con- trolled by the mutant promoter.] RNA polymerase bs ' Nontemplate 3-End of RNA >, being elongated | Positive supercolls Negative supercolls Template strand RNA-DNA hybrid helix Figure 31.8 Local unwinding of DNA by RNA polymerase and formation of an open initiation complex (transcription bubble). Ill. Prokaryotic Gene Transcription 437 2. Elongation: Once the promoter has been recognized and bound by the holoenzyme, local unwinding of the DNA helix continues (Fig. 31.8), mediated by the polymerase. [Note: Unwinding gener- ates supercoils in the DNA that can be relieved by DNA topoisom- erases (see p. 417).] ANA pol begins to synthesize a transcript of the DNA sequence, and several short pieces of RNA are made and discarded. The elongation phase begins when the transcript (typically starting with a purine) exceeds 10 nucleotides in length. Sigma is then released, and the core enzyme is able to leave (clear) the promoter and move along the template strand in a processive manner, serving as its own sliding clamp. During transcription, a short DNA-RNA hybrid helix is formed (see Fig. 31.8). Like DNA pol, RNA pol uses nucleoside 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 ribonucleo- tide causes RNA pol to pause, backtrack, cleave the transcript, and restart. Nonetheless, transcription has a higher error rate than does replication.]. 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 p (rho) factor. a. p-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-comple- mentary (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. b. p-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.. Antibiotics: Some antibiotics prevent bacterial cell growth by inhib- iting RNA synthesis. For example, rifampin (rifampicin) inhibits tran- scription initiation by binding to the 6 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 applica- tion in tumor chemotherapy. It inserts (intercalates) between the DNA bases and inhibits transcription initiation and elongation. DNA coding strand AGCCCGCNNNNNGCGGGCTTTT TCGGGCGNNNNNCGCCCGAAAA DNA template strand J Nascent RNA AGCCCGCNNNNNGCGGGCUUUU 'B) Hatin gv AgAANaPY ee ee Zzanaaaad qa 2m Newly synthesized RNA folds to form a “halrpin’ that Is important in chain termination. Figure 31.9 Rho-independent termination of prokaryotic transcription. A. DNA template sequence generates a self-complementary sequence in the nascent RNA. B. Hairpin structure formed by the RNA. N represents a noncomplementary base; A = adenine, T = thymine; G = guanine; C = cytosine; U = uracil. 438 31. RNA Siructure, Synthesis, and Processing A No drug present FRNA polymerase B| Rifampin present war Rifampin RNA polymerase with distorted conformation Rifampin binds to ANA polymerase and prevents chaln growth beyond three nucleotides. Eukaryotic ANA polymerases do not bind rifampin, and transcription Is unaffected. Figure 31.10 Inhibition of prokaryotic ANA polymerase by rifampin (rifampicin). HCH HHL SN GHS G- SNe GHG Nike fe CH, HAT CH, cH Ss | 2 ———=- | 2 CH, HDAC CH, NH;t NH ae CH, Figure 31.11 Acetylation/deacetylation of a lysine residue in a histone. Acetyl coenzyme A provides the acetyl group. HAT = histone acetyltransferase, HDAC = histone deacetylase. IV. EUKARYOTIC GENE TRANSCRIPTION The transcription of eukaryotic genes is a far more complicated pro- cess 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 po/ 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 decon- densed (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 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 mostinac- tive 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 nucleosomes is also required to access DNA.] 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 |: This enzyme synthesizes the precursor of the 28S, 185, and 5.8S rRNA in the nucleolus. 2. RNA polymerase II: This enzyme synthesizes the nuclear precur- sors of mRNA that are processed and then translated to proteins. RNA pol Ilalso synthesizes certain small ncRNA, such as snoRNA, snRNA, and miRNA. a. Promoters for RNA polymerase Il: In some genes tran- scribed by ANA pol Il, 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 IV. Eukaryotic Gene Transcription 439 Regulatory elements —————_- Distal | Proximal Core promoter elements Enhancer CAAT ec TATA Inr DPE element box box pox +1 +25 Figure 31.12 Eukaryotic gene cis-acting promoter and regulatory elements and their trans-acting general and specific transcription factors (GTF and STF, respectively). Inr = initiator; DPE = downstream promoter element. such as Inr (initiator) or DPE (downstream promoter element) are present (Fig. 31.12). [Note: No one consensus sequence Al is found in all core promoters.] Because these sequences are Direction on the same molecule of DNA as the gene being transcribed, transcription they are cis-acting. The sequences serve as binding sites for wt ———s proteins known as general transcription factors (GTF), which in RVAYIVAVAVAAWAA AY? DNA turn interact with each other and with RNA pol Il. —Core promoter— Transcribed region b. General transcription factors: GTF are the minimal require- f) ments for recognition of the promoter, recruitment of RNA pol DNA bending can cause an enhancer if to the promoter, formation of the preinitiation complex, and element that is far from the promoter initiation of transcription at a basal level (Fig. 31.13A). GTF are with the traneeription initiation encoded by different genes, synthesized in the cytosol, and dif- fuse (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 poly- merase, allowing it to clear the promoter.] complex, stimulating transcription. region 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 Figure 31.13 nucleotides) are the proximal regulatory elements, such as the A. Association of the general CAAT and GC boxes. Those farther away are the distal regulatory transcription factors (TFIl) and elements such as enhancers (see d. below). Proteins known as RNA polymerase II (RNA pol Ii) transcriptional activators or specific transcription factors (STF) at the core promoter. [Note: The bind these regulatory elements. STF bind to promoter proximal Fee er RNA col Il] B Enheneer elements to regulate the frequency of transcription initiation and to stimulation of transcription. distal elements to mediate the response to signals such as hor- CTF = CAAT box transcription mones (see p. 472) and regulate which genes are expressed ai a factor; Sp1 = specificity factor-1. 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 activa- tion domain that recruits the GTF to the core promoter as well as coactivator proteins such as the HAT enzymes involved in 440 31. RNA Structure, Synthesis, and Processing ‘An enhancer sequence can be chromatin modification. [Note: Mediator, a multisubunit coactivator upstream from the promoter region. | of RNA pol /Fcatalyzed transcription, binds the polymerase, the v GTF, and the STF and regulates transcription initiation.] DNA »*) 5’-| Enhancer|- s;—{ P [70% of individuals with CF, the AF508 mutation is the cause of the disease. lil. 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 ter- mination 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 miss- ing, 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 contin- ued protein synthesis. ] Ill. Components Required for Translation 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. 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 link- age 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 iRNA 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 car- ried by that tRNA.. Aminoacyl-tRNA synthetases 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 cor- respond 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 «-carboxyl group of an amino acid to the Ain the -CCA sequence at the 3’-end of its corresponding tRNA. The overall reaction requires ATP, which is cleaved to adenos- ine monophosphate and inorganic pyrophosphate (PPi), as shown in Figure 32.7. The extreme specificity of the synthetases in recogniz- ing both the amino acid and its cognate tRNA contributes to the high fidelity of translation of the genetic message. In addition to their syn- thetic 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.. 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.]. 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. 451 Amino acid Aminoacy!-tRNA fouls synthetase (E) PP| —> 2P| E-AMP~Amino acid CCA AMP E CA~Amino acid Aminoacyl-tRNA Figure 32.7 Attachment of a specific amino acid to its corresponding transfer RNA (tRNA) by an aminoacyl-tRNA synthetase. PPj = pyrophosphate; P; = inorganic phosphate; A = adenine; C = cytosine; AMP = adenosine monophosphate; ~ = high-energy bond. 452 32. Protein Synthesis PROKARYOTIC RIBOSOME 23S RNA 16S RNA A 33 Proteins 21 Proteins For example, the prokaryotic 50S and 30S ribosomal subunits together form a 70S ribosome. The eukaryotic 60S and 40S sub- units 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. 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. aS 28S RNA 18S RNA Vv ~50 Proteins ~35 Proteins Figure 32.8 Ribosomal composition. [Note: The number of proteins in the eukaryotic ribosomal subunits varies somewhat from species to species.] S = Svedberg unit. 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. 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 ribo- some. (See Fig. 32.13 for an illustration of the role of the A, P, and E sites in translation.) 4. Cellular location: In eukaryotic cells, the ribosomes either are free in the cytosol or are in close association with the endoplas- mic 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 mem- branes, 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, mito- chondria, 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.] IV. Codon Recognition by Transfer RNA 453 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 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 PP; and one in the subsequent hydrolysis of the PPi, to two Pj 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 ini- tiation 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 (isoac- cepting tRNA) recognize more than one codon for a given amino acid. 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 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. Serine ACC sSSEE Complementary (antiparallel) binding Anticodon (5-UGA-3) Nontraditional base-palring possible between Traditional base- pairing observed In first and second positions of codon:|] the third (3) tRNA mRNA poser orine (A__U) codon and the (ec) first (54 position of the anticodon: (CG) tRNA mRNA Figure 32.9 Wobble: Nontraditional base-pairing between the 5’-nucleotide (first nucleotide) of the anticodon and the 3’-nucleotide (last nucleotide) of the codon. Hypoxanthine (H) is the product of adenine deamination and the base in the nucleotide inosine monophosphate (IMP). A = adenine; G = guanine; C = cytosine; U = uracil; tRNA = transfer RNA; mRNA = messenger RNA. 454 32. Protein Synthesis 16S Ribosomal RNA (rRNA) ) 3-End \ Y-End ; Y |} Messenger RNA (mRNA) yh YUCCUCC TA 5-End “PUAAGGAGG AUG Surg-End Shine-Dalgarno sequence 30S Ribosomal subunit Figure 32.10 Complementary binding between prokaryotic mRNA Shine-Dalgarno sequence and 16S rRNA. S = Svedberg unit. [@)_V. STEPS IN TRANSLATION The process of protein synthesis translates the 3-letter alphabet of nucle- — otide sequences on mRNA into the 20-letter alphabet of amino acids that i constitute proteins. The mRNA is translated from its 5’-end to its 3’-end, pe producing a protein synthesized from its amino (N)-terminal end to its pre carboxyl (C)-terminal end. Prokaryotic mRNA often have several coding CCE ta Nie regions (that is, they are polycistronic; see p. 434). Each coding region coat has its own initiation and termination codon and produces a separate ernionine 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. Initlator tRNA (tRNA) One important difference is that translation and transcription N!°.Formyl-THF Tenens are temporally linked in prokaryotes, with translation starting before transcription is completed as a consequence of the CHs lack of a nuclear membrane in prokaryotes. THF $s CHa ata CHp a) A. Initiation Gee Ne Initiation of protein synthesis involves the assembly of the compo- OH nents of the translation system before peptide-bond formation occurs. Nrormyt 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 assem- bly 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 elF 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. fMet-tRNA, Figure 32.11 1. Shine-Dalgarno sequence: In Escherichia coli (E. coli), a purine- Generation of the initiator rich sequence of nucleotide bases, known as the Shine-Dalgarno N-formylmethionyl-transfer RNA (fMet-tRNA)). THF = tetrahydrofolate; C = cytosine; A = adenine. (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 V. Steps in Translation 455 16S rRNA component of the small (80S) 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 Peptide chain 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. 5'-Cap: Eukaryotic mRNA do not have SD sequences. In eukary- otes, the small (40S) ribosomal subunit (aided by members of the elF-4 family of proteins) binds close to the cap structure at the 5’-end of the mRNA and moves 5’—>3’ along the mRNA unitil 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 elF-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. Initiation codon: The initiating AUG is recognized by a special initiator tRNA (tRNA\). Recognition is facilitated by IF-2-GTP in pro- karyotes and elF-2-GTP (plus additional elF) in eukaryotes. The charged tRNAji is the only tRNA recognized by (e)IF-2 and the only transferase tRNA to go directly to the P site on the small subunit. [Note: Base (ribozyme) modifications distinguish tRNA; 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 transformyiase, which uses N'°-formyl tetrahydrofolate (see p. 267) as the carbon donor. In eukaryotes, tRNA; 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 tRNA; 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 elF-2B facilitates the reactivation of elF-2-GDP through replacement of GDP by GTP. B. Elongation 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 elon- gation factors EF-Tu-GTP and EF-Ts and requires GTP hydrolysis. [Note: In eukaryotes, comparable elongation factors are EF-10-GTP and EF-1fy. Both EF-Ts and EF-1y function in guanine nucleotide exchange.] Peptide-bond formation between the «-carboxyl group of Figure 32.12 the amino acid in the P site and the «-amino group of the amino acid Formation of a peptide bond. Peptide- in the A site is catalyzed by peptidyitransferase, an activity intrinsic to bond formation results in transfer of the an rRNA of the large subunit (Fig. 32.12). [Note: Because this rRNA Peptide on the transfer RNA (tRNA) catalyzes the reaction, it is a ribozyme (see p. 54).] After the peptide in the P site to the amino acid on the bond has been formed, the peptide on the tRNA at the P site is trans- tRNA in the A site (transpeptidation).. i. mRNA = messenger RNA; R’, R’ = ferred to the amino acid on the tRNA at the A site, a process known different amino aad side chains. as transpeptidation. The ribosome then advances three nucleotides 456 32. Protein Synthesis STREPTOMYCIN binds to the 30S subunit and distorts Its structure, Interfering with the initiation of protein synthesis. subunit arrives to form the 70S initiation complex. IF-1 IF=-3 AAG---CGGUAA~™™ 3 IF-3 mRNA /IF-2-GTP IF-2-GDP +P, P site Initlation factors (IFs) ald In the| formation of the 30S initiation complex. The charged initiator tRNA Is brought to the P site of the 30S subunit by IF-2-GTP. TETRACYCLINES Qe Phenylalanyl-tRNA Interact with the 30S subunit, Perera | EF-Tu-GTP. blocking access of the q ‘) EF-Ts aminoacyl-tRNA to the A site, Elongation factor EF-Tu-GTP EF-Tu-GDP GTP thereby Inhibiting elongation. brings the appropriate, charged | tRNA to the codon in the empty A slte (decoding). The GTP Peptide bond is hydrolyzed to GDP. EF-Ts mediates exchange of GDP for GTP. Asite ELONGATION Peptidyltransferase, an activity of |/ the 23S rRNA of the 50S subunit, catalyzes peptide-bond formation, transferring the initiating amino acid (or growing peptide chain) from the P site to the amino acid at the A site (transpeptidation). Peptidyl- transferase PUROMYCIN bears a structural resemblance to aminoacyl-tRNA and accepts a peptide from the P site, causing inhibition of elongation and resulting in premature termination in both prokaryotes and eukaryotes. CHLORAMPHENICOL inhibits prokaryotic peptidyltransferase. High levels may also Inhibit mitochondrial protein synthesis. Figure 32.13 (continued on next page) Steps in prokaryotic protein synthesis (translation), and their inhibition by antibiotics. [Note: EF-Ts is a guanine nucleotide exchange factor. It facilitates the removal of guanosine diphosphate (GDP) from EF-Tu, allowing its replacement by guanosine triphosphate (GTP). The eukaryotic equivalent is EF-1By.] fMet = formylated methionine; S = Svedberg unit; Phe = phenylalanine; Lys = lysine; Arg = arginine; tRNA = transfer RNA; mRNA = messenger RNA. V. Steps in Translation 457 EF-G-GTP facilitates movement of the ribosome three nucleotides along the mRNA In the 53’ direction. What was In the P site Is now In E, what was In the A site is now in P, and A is empty. GTP Is hydrolyzed to GDP. EF-G-GTP EF-G-GDP +P, MeL Pa — x ,: Translocation \ _ ERYTHROMYCIN binds irreversibly to a site on the 50S subunit and blocks the tunnel by which the peptide leaves the rlbosome, thereby Inhibiting translocation. Steps 3, 4, and 5 are repeated until a termination codon is encountered at the A site. Termination codon 7 A termination codon Is recognized by a release factor (RF-1 or RF-2), which results in release of the newly synthe- sized protein. GTP on RF-3 Is hydrolyzed. The synthesizing complex dissociates. Completed peptide a Figure 32.13 (continued from previous page) [Note: In eukaryotes, diphtheria toxin inactivates EF-2, thereby inhibiting the translocation phase of elongation. Ricin, a toxin from castor beans, removes a specific A from the 28S ribosomal RNA (rRNA) in the large subunit of eukaryotic ribosomes, thereby inhibiting ribosomal function.] 458 32. Protein Synthesis Cell Factor Function P IF-2-GTP | Bring charged initiat- elF-2-GTP ing tRNA to P site P IF-3 Prevent association | elF-3 of subunits Bring all other P | EF-Tu-GTP E | EFla-GTp | charged tRNA to Asite P EF-Ts Guanine nucleotide E EF-1By exchange factors P EF-G-GTP. E EF-2-GTP Translocation P RF-1, 2 Recognize stop E eRF codons P RF-3-GTP eRF-3-GTP Release of other RF Figure 32.14 Protein factors in the three stages of translation. P = prokaryotes; E = eukaryotes; tRNA = transfer RNA; IF = initiation factor; EF = elongation factor; RF = release factor; GTP = guanosine triphosphate. toward the 3’-end of the mRNA. This process is known as translo- cation 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.]. Termination 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 rec- ognizes 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, 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.] & = 80S Abscess on messenger RNA N-Terminal signal sequence on peptide ———> NH, : Ctr CYTOSOL RER MEMBRANE Oe RER LUMEN Pp) dj SRP Translocon Translocon aoe Dae (closed) (open) cleaved by signa Receptor peptidase NH, Figure 32.15 Cotranslational targeting of proteins to the rough endoplasmic reticulum (RER). SRP = signal recognition particle. VI. Co- and Posttranslational Modifications 459 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 elF-2: Phosphorylated elF-2 is inactive (see p. 476). In both eukary- otes and prokaryotes, regulation can also be achieved through pro- teins 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 Although most protein synthesis in eukaryotes is initiated in the cyto- plasm, many proteins perform their functions within subcellular organ- elles 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 (cotransla- tional targeting) to the RER by the presence of an N-terminal hydro- phobic signal sequence. The sequence is recognized by the signal recognition particle (SRP), a ribonucleoprotein that binds the ribo- some, 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 (posttrans- lational) include nuclear proteins that contain an internal, short, basic nuclear localization signal; mitochondrial matrix proteins that contain an N-terminal, amphipathic, «-helical mitochondrial entry sequence; and peroxisomal proteins that contain a C-terminal tripeptide signal. 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, pre- cursor molecules that are not functionally active. Portions of the pro- tein must be removed by specialized endoproteases, resulting in the release of an active molecule. The cellular site of the cleavage reac- tion 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 Protein function can be affected by the covalent attachment of a vari- ety of chemical groups (Fig. 32.16). Examples include the following. Phosphorylation Phosphate 3 — ~ ~O-P-O—CHp- CH 1 o , NH Serine g Protein - G50 fo > CH2- CH o J NH Tyrosine Glycosylation ~ 9 CH, 0. HO H on u , 2° O-CH,- CH Serine 1 NH NH ¢=0 a N-Acetyl- galactosamine CH,0OH (eo) c=-0 © NH-G-CHp-CH H a a ae 0 OH H H NH Asparagine 2 NH ¢-0 in N-Acetyl- glucosamine Figure 32.16 (continued on next page) Covalent modification of some amino acid residues. 460 32. Protein Synthesis 1. Phosphorylation: Phosphorylation occurs on the hydroxyl groups of serine, threonine, or, less frequently, tyrosine residues in 7 a protein. It is catalyzed by one of a family of protein kinases and | may be reversed by the action of protein phosphatases. The phos- rg oes ee phorylation may increase or decrease the functional activity of the H2C. _CH, protein. Several examples of phosphorylation reactions have been CH | - Hydroxyprolyl residue Carboxylation Mature clotting factors anon N- CH-Geens VII, IX, X Gt.e) -Carboxyglutamyl a coo: | © Gla) residue i Biotinylated enzyme H ah rnar NO CH—-C AAA CH, O Lysyl residue —> fae of a carboxylase CH, vn NH ie) oO HN“ NH Biotin Blotin-enzyme Farnesylated protein Cysteine ; a 0-6 CH; CH2- S-CHy-CH CH, CH, C= o e NH CH, Slt, C= Cc CH, ‘c-c cH, 4H cH, -H eee Farnesyl group Figure 32.16 (continued from previous page) Covalent modification of some amino acid residues. previously discussed (for example, see Chapter 11, p. 132, for the regulation of glycogen synthesis and degradation). 2. Glycosylation: Many of the proteins that are destined to become part of a membrane or to be secreted from a cell have carbohy- drate chains added en bloc to the amide nitrogen of an aspara- gine (N-linked) or built sequentially on the hydroxyl groups of a serine, threonine, or hydroxylysine (O-linked). N-glycosylation occurs in the RER and O-glycosylation in the Golgi. (The pro- cess of producing such glycoproteins was discussed on p. 165.) N-glycosylated acid hydrolases are targeted to the matrix of lyso- somes by the phosphorylation of mannose residues at carbon 6 (see p. 169). 3. Hydroxylation: Proline and lysine residues of the « 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-depen- dent carboxylation (see p. 393). The resulting y-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 e-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 influ- ences gene expression (see p. 476).] 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 macromo- lecular, 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 pro- teins 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 elF-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 461 i > Vil. CHAPTER SUMMARY 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 mutatlons (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 to be synthesized; fully competent ribosomes (70S in prokaryotes, 80S in eukaryotes); proteln 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 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 complementary sequence on 16S rRNA, resulting in the positioning of the small subunit on the mRNA so that translation can begin. The 5’-eap (bound by proteins of the elF-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 (tRNA)) 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 peptidyltransferase, which is an activity intrinsic to the rRNA of the large subunit and, therefore, is a rlbozyme. 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 antiblotles 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 cytosolic complex known as the proteasome. J 462 32. Protein Synthesis Flow of genetic information [DNA is an _| Informational | composed of Sequence of visualized as molecule deoxyribonucleotides provides Information transfer by ¥ Transcription visualized as resulting in the synthesis of [ mRNA | is an ,| Informational composed of Sequence of visualized as molecule ribonucleotides provides Information transfer by e Specific Amino acid eo Universal ‘N\ Triplet codons defined by , Genetic code characterized as e Degenerate e Nonoverlapping e Commaless which pair with Specific anticodons | Aminoacy|-tRNA / “ in A specific tRNA \ Anticodon —> UAC recognizes iF AUG errr Aml I- synthesized bi visualized as a RNA "tRNA ” v Aminoacy-tRNA| Codon synthetase _| deliver amino recognizes acids to Bast A specific amino acld visualized as [ Ribosomes | SE PSEEE GE One small and rT one large subunit resulting in synthesis of = isa ,| Functional | consistingof | Sequenceof | visualized as Protein molecule amino acids Figure 32.17 Key concept map for protein synthesis. mRNA = messenger RNA; tRNA = transfer RNA; A = adenine; G = guanine; C = cytosine; U = uracil. VII. Chapter Summary 463 Study Questions Choose the ONE best answer. 32.1 32.2 32.3 32.4 A 20-year-old man with a microcytic anemia is found to have an abnormal form of 6-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. CGA > UGA GAU > GAC GCA > GAA UAA > CAA UAA > UAG moOD> 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 Asite C. Peptidyltransferase activity D. Ribosomal translocation E. Termination A transfer RNA (tRNA) molecule that is supposed to carry cysteine (tRNA™*) is mischarged, so that it actually carries alanine (ala-tRNA™*). 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. Ina patient with cystic fibrosis (CF) caused by the AF508 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. It is degraded by the proteasome. It is placed into storage vesicles. It is repaired by cellular enzymes. It is secreted from the cell. moow ™ Correct answer = D. Mutating the normal termination (stop) codon from UAA to CAA in f-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 pro- tein. 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. Correct answer = D. Because fMet-Phe (formylated methi- onyl-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. a Correct answer = B. Once an amino acid is attached to a tRNA molecule, only the anticodon of that tRNA deter- mines the specificity of incorporation. Therefore, the incorrectly activated alanine will be incorporated into the protein at a position determined by a cysteine codon. Correct answer = B. Ubiquitination usually marks old, damaged, or misfolded proteins for destruction by the cytosolic proteasome. There is no known cellular mecha- nism for repair of damaged proteins. 464 32. Protein Synthesis 32.5 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. C. Streptomycin binds to the 30S ribosomal subunit. D. Tetracyclines inhibit peptidyltransferase. 32.6 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? AAC ACA CAA CAC CCA moOW> 32.7 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 32.8 a1-Antitrypsin (AAT) deficiency can result in emphy- sema, 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 endo- plasmic 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. 32.9 Why is the genetic code described as both degenerate and unambiguous? 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 ami- noacyl-transfer RNA. It is incorporated into the growing chain, inhibits elongation, and results in premature termi- nation in both prokaryotes and eukaryotes. Tetracyclines bind the 30S ribosomal subunit and block access to the A site, inhibiting elongation. X f 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. f 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 postiranscriptionally process their mRNA. 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 ribo- some, 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 pack- aged 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 man- nose residues in glycoproteins targets these proteins (acid hydrolases) to lysosomes. The N-terminal methio- nine is removed from most proteins during processing. & 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). 4 Regulation of Gene Expression Il. 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, how- ever, 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 prod- ucts that are produced. 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 prokary- otes, but many themes are repeated in eukaryotes. Figure 33.1 shows some of the sites where gene expression can be controlled. ll. REGULATORY SEQUENCES AND MOLECULES Regulation of transcription, the initial step in all gene expression, is con- trolled 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 For most genes, the main site mRNA { Transcription Protein ! Translation Transcription Primary RNA transcript —EEEs te Proteins SS = lodifled proteins! == = In eukaryotes, gene expression Is also controlled at posttranscriptional and positranslatlonal processes. Figure 33.1 Control of gene expression. MRNA = messenger RNA. 465 466 33. Regulation of Gene Expression Trans-acting factors, usually proteins, are synthesized from genes that are different from the genes targeted for regulation. Trans-acting factors bind to cis-acting elements on DNA. < Trans-acting factor él : Direction i seariet Nictton { Pol fi nr DNA Transcribed region | Cis-acting elements s! Cls-acting elements are DNA sequences that are bound by trans- acting regulatory factors. 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), leu- cine zipper, or helix-turn-helix in the protein. lll. REGULATION OF PROKARYOTIC GENE EXPRESSION Figure 33.2 Cis-acting elements and trans-acting factors. mRNA = messenger RNA; Po/ ll = RNA polymerase Il. Two antiparallel B-strands f-turn Figure 33.3 Zine (Zn) finger is a common motif in proteins that bind DNA. Cys = cysteine; His = histidine. In prokaryotes such as the bacterium Escherichia coli (E. coli), regu- lation 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 prod- ucts.] Transcriptional control in prokaryotes can involve the initiation or premature termination of transcription. 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 Bacterial operons contain an operator, a segment of DNA that regu- lates 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 pro- teins) 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 po can initi- ate transcription. One of the best-understood examples is the induc- ible lactose (lac) operon of E. coli that illustrates both positive and negative regulation (Fig. 33.4). C. Lactose operon The lac operon contains the genes that code for three proteins involved in the catabolism of the disaccharide lactose: the lacZ gene codes for -galactosidase, 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 Ill. Regulation of Prokaryotic Gene Expression 467 | + Glucose —Lactose Operon repressed (off) | @ CAP tet Gunbound) lac Adenylyl cyclase is inactive in the presence of glucose, 7 \ VS mi d CAP is not —>— mRNA , “ty bound to CAMP: uy y — (i catabolite repression. | A “ | \\ r Repressor Oy Pa \ Transcription Is No mRNA and, protein prevented by the therefore, no proteins repressor protein. are produced.* Operator is not blocked, and the CAP site is occupied. RNA polymerase can efficiently initiate transcription. GB) = Sses? Operon induced (on) —— a —”. “a val Q) 2 cAMP i122 mRNA ————= \/ ¢