Biochemistry, 4th Edition P96 pps

29.2 How Is Transcription Regulated in Prokaryotes? 913 transcribed determines whether transcription takes place. This sequence is termed the operator (Figure 29.8). The operator is located next to a promoter. Interaction of a regulatory protein with the operator controls transcription of the gene cluster by con- trolling access of RNA polymerase to the promoter. 3 Such co-expressed gene clusters, together with the operator and promoter sequences that control their transcription, are called operons. Transcription of Operons Is Controlled by Induction and Repression In prokaryotes, gene expression is often responsive to small molecules serving as sig- nals of the nutritional or environmental conditions confronting the cell. Increased synthesis of enzymes in response to the presence of a particular substrate is termed (a) RNA polymerase ter site mRNA ␳ factor (b) (c) (d) mRNA FIGURE 29.7 The rho factor mechanism of tran- scription termination. Rho factor (a) attaches to a recognition site on mRNA and (b) moves along it behind RNA polymerase. (c) When RNA polymerase pauses at the termination site, rho factor unwinds the DNAϺRNA hybrid in the transcription bubble, (d) releasing the nascent mRNA. DNA O p Transcriptional control region Operator Promotor 123 Structural genes 1, 2, 3 FIGURE 29.8 The general organization of operons. Operons consist of transcriptional control regions and a set of related structural genes, all organized in a contiguous linear array along the chromosome.The transcrip- tional control regions are the promoter and the operator, which lie next to, or overlap, each other, upstream from the structural genes they control. Operators may lie at various positions relative to the promoter, either upstream or downstream. Expression of the operon is determined by access of RNA polymerase to the pro- moter, and occupancy of the operator by regulatory proteins influences this access. Induction activates tran- scription from the promoter; repression prevents it. 3 Although this is the paradigm for prokaryotic gene regulation, it must be emphasized that many prokaryotic genes do not contain operators and are regulated in ways that do not involve proteinϺop- erator interactions. 914 Chapter 29 Transcription and the Regulation of Gene Expression induction. For example, lactose (Figure 29.9) can serve as both carbon and energy source for E. coli. Metabolism of lactose depends on hydrolysis into its component sug- ars, glucose and galactose, by the enzyme ␤-galactosidase. In the absence of lactose, E. coli cells contain very little ␤-galactosidase (less than 5 molecules per cell). How- ever, lactose availability induces the synthesis of ␤-galactosidase by activating transcrip- tion of the lac operon. One of the genes in the lac operon, lacZ, is the structural gene for ␤-galactosidase. When its synthesis is fully induced, ␤-galactosidase can amount to almost 10% of the total soluble protein in E. coli. When lactose is removed from the culture, synthesis of ␤-galactosidase halts. The alternative to induction—namely decreased synthesis of enzymes in response to a specific metabolite—is termed repression. For example, the enzymes of tryptophan biosynthesis in E. coli are encoded in the trp operon. If sufficient Trp is available to the growing bacterial culture, the trp operon is not transcribed, so the Trp biosynthetic enzymes are not made; that is, their synthesis is repressed. Repression of the trp operon in the presence of Trp is an eminently logical control mechanism: If the end product of the pathway is present, why waste cellular resources making unneeded enzymes? Induction and repression are two faces of the same phenomenon. In induction, a substrate activates enzyme synthesis. Substrates capable of activating synthesis of the enzymes that metabolize them are called co-inducers, or, often, simply inducers. Some substrate analo gs can induce enzyme synthesis even though the enzymes are incapable of metabolizing them. These analogs are called gratuitous inducers. A number of thiogalactosides, such as IPTG (isopropyl ␤-thiogalactoside, Figure 29.10), are excellent gratuitous inducers of ␤-galactosidase activity in E. coli. In re- pression, a metabolite, typically an end product, depresses synthesis of its own biosynthetic enzymes. Such metabolites are called co-repressors. The lac Operon Serves as a Paradigm of Operons In 1961, François Jacob and Jacques Monod proposed the operon hypothesis to ac- count for the coordinate regulation of related metabolic enzymes. The operon was considered to be the unit of gene expression, consisting of two classes of genes: the structural genes for the enzymes and regulatory genes that control expression of the structural genes. The two kinds of genes could be distinguished by mutation. Muta- tions in a structural gene would abolish one particular enzymatic activity, but muta- tions in a regulatory gene would affect all of the different enzymes under its control. Mutations of both kinds were known in E. coli for lactose metabolism. Bacteria with mutations in either the lacZ gene or the lacY gene (Figure 29.11) could no longer metabolize lactose—the lacZ mutants (lacZ Ϫ strains) because ␤-galactosidase activity CH 2 OH O HO OH O OH CH 2 OH O OH OH OH Lactose (O-␤- D-galactopyranosyl (1 4) ␤-D-glucopyranose) FIGURE 29.9 The structure of lactose, a ␤-galactoside. CH 2 OH O HO OH S OH CH CH 3 CH 3 Isopropyl ␤-thiogalactoside (IPTG) FIGURE 29.10 The structure of IPTG (isopropyl ␤-thiogalactoside). DNA Polypeptide p Amino acids kD Protein Structure kD Function 360 38.6 Tetramer 154.4 Repressor 1023 116.4 Tetramer 465 ␤-Galactosidase 417 46.5 46.5 Permease 203 22.7 Dimer 45.4 Trans- acetylase Membrane protein mRNA bp 1080 3069 1251 60982 lacI p lac ,O lacZ lacY lacA FIGURE 29.11 The lac operon.The operon consists of two transcription units. In one unit, there are three structural genes, lacZ, lacY, and lacA, under control of the promoter,p lac , and the operator O. In the other unit, there is a regulator gene, lacI, with its own promoter, p lacI . lacI encodes a 360-residue, 38.6-kD polypeptide that forms a tetrameric lac repressor protein. lacZ encodes ␤-galactosidase, a tetrameric enzyme of 116-kD subunits. lacY is the ␤-galactoside permease structural gene, a 46.5-kD integral membrane protein active in ␤-galactoside transport into the cell.The remaining structural gene encodes a 22.7-kD polypeptide, but the metabolic role of this protein in vivo remains uncertain. 29.2 How Is Transcription Regulated in Prokaryotes? 915 was absent, the lacY mutants because lactose was no longer transported into the cell. Other mutations defined another gene, the lacI gene. lacI mutants were different be- cause they both expressed ␤-galactosidase activity and immediately transported lac- tose, without prior exposure to an inducer. That is, a single mutation led to the expres- sion of lactose metabolic functions independently of inducer. Expression of genes independently of regulation is termed constitutive expression. Thus, lacI had the properties of a regulatory gene. The lac operon includes the regulatory gene lacI; its promoter p; and three structural genes, lacZ, lacY, and lacA, with their own promoter p lac and operator O (Figure 29.11). lac Repressor Is a Negative Regulator of the lac Operon The structural genes of the lac operon are controlled by negative regulation. That is, they are transcribed to give an mRNA unless turned off by the lacI gene product. This gene product is the lac repressor, a tetrameric protein (Figure 29.12). (Note that the language can be misleading: Inducers and co-repressors are p lac p lac mRNA DNA lacI O lacZ lacY lacA No transcription Repressor monomer Repressor tetramer mRNA DNA lacI O lacZ lacY lacA Transcription Repressor monomer Repressor tetramer Inducer mRNA Translation ␤-Galactosidase Permease Transacetylase Without inducer With inducer FIGURE 29.12 The mode of action of lac repressor.The structure of the lac repressor tetramer with bound IPTG (purple) is also shown (pdb id ϭ 1LBH). 916 Chapter 29 Transcription and the Regulation of Gene Expression small molecules/metabolites; repressors are proteins.) The lac repressor has an N-terminal DNA-binding domain; the rest of the protein functions in inducer binding and tetramer formation. In the absence of an inducer, lac repressor blocks lac gene expression by binding to the operator DNA site upstream from the lac structural genes. The lac operator is a palindromic DNA sequence (Figure 29.13). Palindromes, or “inverted repeats,” provide a twofold, or dyad, symmetry, a structural feature common at sites in DNA where proteins specifically bind. Despite the presence of lac repressor, RNA polymerase can still initiate tran- scription at the promoter (p lac ), but lac repressor blocks elongation of transcrip- tion, so initiation is aborted. In lacI mutants, the lac repressor is absent or defec- tive in binding to operator DNA, lac gene transcription is not blocked, and the lac operon is constitutively expressed in these mutants. Note that lacI is normally expressed constitutively from its promoter, so lac repressor protein is always avail- able to fill its regulatory role. About ten molecules of lac repressor are present in an E. coli cell. Derepression of the lac operon occurs when appropriate ␤-galactosides occupy the inducer site on lac repressor, causing a conformational change in the protein that lowers the repressor’s affinity for operator DNA. As a tetramer, lac repressor has four inducer binding sites, and its response to inducer shows cooperative allosteric effects. Thus, as a consequence of the “inducer”-induced conformational change, the inducerϺlac repressor complex dissociates from the DNA, and RNA polymerase transcribes the structural genes (see Figure 29.12). Induction reverses rapidly: lac mRNA has a half-life of only 3 minutes, and once the inducer is used up through metabolism by the enzymes, free lac repressor reassociates with the operator DNA, transcription of the operon is halted, and any residual lac mRNA is degraded. In the absence of inducer, lac repressor binds nonspecifically to duplex DNA with an association constant, K A , of 2 ϫ 10 6 M Ϫ1 (Table 29.1) and to the lac operator DNA sequence with much higher affinity, K A ϭ 2 ϫ 10 13 M Ϫ1 . Thus, lac repressor binds 10 7 times better to lac operator DNA than to any random DNA se- quence. IPTG binds to lac repressor with an association constant of about 10 6 M Ϫ1 . The IPTGϺlac repressor complex binds to operator DNA with an association con- stant, K A ϭ 2 ϫ 10 10 M Ϫ1 . Although this affinity is high, it is 3 orders of magnitude less than the affinity of inducer-free repressor for lac operator. There is no differ- ence in the affinity of free lac repressor and lac repressor with IPTG bound for non- operator DNA. The lac repressor apparently acts by binding to DNA and sliding along it, testing sequences in a one-dimensional search until it finds the lac opera- tor. The lac repressor then binds there with high affinity until inducer causes this affinity to drop by 3 orders of magnitude (Table 29.1). CAP Is a Positive Regulator of the lac Operon Transcription by RNA polymerase from some promoters proceeds with low efficiency unless assisted by an accessory protein that acts as a positive regulator. One such protein is CAP, or catabolite activator protein. Its name derives from the phenomenon of catabolite repression in E. coli. Catabolite repression is a global control that coordi- A T T A G C T A G C A T T A T A C G A T DNA G C T A G C T A T A G C T A G C A T G C T A A T A T C G A T A T T A T A T A C G A T C G A T C G A T G C G C –10 –5 +1 +5 +10 +15 +20 +25 Axis of symmetry Protected bases mRNA transcription G C TT T AG AT A GG A T G TTG A T A T TTTG FIGURE 29.13 The nucleotide sequence of the lac oper- ator.This sequence comprises 36 bp showing nearly palindromic symmetry.The inverted repeats that consti- tute this approximate twofold symmetry are shaded in rose.The bases are numbered relative to the ϩ1 start site for transcription.The GϺC base pair at position ϩ11 represents the axis of symmetry. In vitro studies show that bound lac repressor protects a 26-bp region from Ϫ5 to ϩ21 against nuclease digestion. Bases that inter- act with bound lac repressor are indicated below the operator. Note the symmetry of protection at ϩ1 through ϩ4 TTAA to ϩ18 through ϩ21 AATT. Repressor ϩ DNA Repressor Inducer lac operator 2 ϫ 10 13 M Ϫ1 2 ϫ 10 10 M Ϫ1 All other 2 ϫ 10 6 M Ϫ1 2 ϫ 10 6 M Ϫ1 DNA Specificity† 10 7 10 4 *Values for repressorϺDNA binding are given as association constants, K A , for the formation of DNAϺrepressor complex from DNA and repressor. †Specificity is defined as the ratio (K A for repressor binding to operator DNA)/(K A for repressor binding to random DNA). TABLE 29.1 The Affinity of lac Repressor for DNA* 29.2 How Is Transcription Regulated in Prokaryotes? 917 nates gene expression with the total physiological state of the cell: As long as glucose is available, E. coli catabolizes it in preference to any other energy source, such as lac- tose or galactose. Catabolite repression ensures that the operons necessary for me- tabolism of these alternative energy sources, that is, the lac and gal operons, remain repressed until the supply of glucose is exhausted. Catabolite repression overrides the influence of any inducers that might be present. Catabolite repression is maintained until the E. coli cells become starved of glu- cose. Glucose starvation leads to activation of adenylyl cyclase, and the cells begin to make cAMP. (In contrast, glucose uptake is accompanied by deactivation of adenylyl cyclase.) The action of CAP as a positive regulator is cAMP-dependent. cAMP is a small-molecule inducer for CAP, and cAMP binding enhances CAP’s affinity for DNA. CAP, also referred to as CRP (for cAMP receptor protein), is a dimer of iden- tical 22.5-kD polypeptides. The N-terminal domains bind cAMP; the C-terminal do- mains constitute the DNA-binding site. Two molecules of cAMP are bound per dimer. The CAP–(cAMP) 2 complex binds to specific target sites near the promoters of operons (Figure 29.14). Binding of CAP–(cAMP) 2 to DNA causes the DNA to bend more than 80° (Figure 29.15). This CAP-induced DNA bending near the pro- moter assists RNA polymerase holoenzyme binding and closed promoter complex formation. Contacts made between the CAP–(cAMP) 2 complex and the ␣-subunit of RNA polymerase holoenzyme activate transcription. Negative and Positive Control Systems Are Fundamentally Different Negative and positive control systems operate in fundamentally different ways (al- though in some instances both govern the expression of the same gene). Genes un- der negative control are transcribed unless they are turned off by the presence of a repressor protein. Often, transcription activation is merely the release from nega- tive control. In contrast, genes under positive control are expressed only if an active regulator protein is present. The lac operon illustrates these differences. The action of lac repressor is negative. It binds to operator DNA and blocks transcription; ex- pression of the operon occurs only when this negative control is lifted through the release of the repressor. In contrast, regulation of the lac operon by CAP is positive: Transcription of the operon by RNA polymerase is stimulated by CAP’s action as a positive regulator. A DEEPER LOOK Quantitative Evaluation of lac RepressorϺDNA Interactions The affinity of lac repressor for random DNA ensures that essentially all repressor is DNA bound. Assume that E. coli DNA has a single spe- cific lac operator site for repressor binding and 4.64 ϫ 10 6 base pairs and any nucleotide sequence even one base out of phase with the operator constitutes a nonspecific binding site. Thus, there are 4.64 ϫ 10 6 nonspecific sites for repressor binding. The binding of repressor to DNA is given by the association constant, K A : K A ϭ where [repressorϺDNA] is the concentration of repressorϺDNA complex, [repressor] is the concentration of free repressor, and [DNA] is the concentration of nonspecific binding sites. Rearrang- ing gives the following: ϭ 1 ᎏᎏ K A [DNA] [repressor] ᎏᎏ [repressorϺDNA] [repressorϺDNA] ᎏᎏᎏ [repressor][DNA] If the number of nonspecific binding sites is 4.64 ϫ 10 6 , there are (4.64 ϫ 10 6 )/(6.022 ϫ 10 23 ) ϭ 0.77 ϫ 10 Ϫ17 moles of binding sites contained in the volume of a bacterial cell (roughly 10 Ϫ15 liters). Therefore, [DNA] ϭ (0.77 ϫ 10 Ϫ17 )/(10 Ϫ15 ) ϭ 0.77 ϫ 10 Ϫ2 M. Since K A ϭ 2 ϫ 10 6 M Ϫ1 (Table 29.1), ϭϭ So, the ratio of free repressor to DNA-bound repressor is 6.5 ϫ 10 Ϫ5 . Less than 0.01% of repressor is not bound to DNA! The behavior of lac repressor is characteristic of DNA-binding proteins. These proteins bind with low affinity to random DNA sequences, but with much higher affinity to their unique target sites (Table 29.1). 1 ᎏᎏ (1.54 ϫ 10 4 ) 1 ᎏᎏᎏ (2 ϫ 10 6 ) (0.77 ϫ 10 Ϫ2 ) [repressor] ᎏᎏ [repressor:DNA] DNA binding and transcriptional activation 72 89 74 89 61 67 72 61 50 79 100 94 55 78 Binding region Upstream of RNA polymerase binding site at –41 or –61 or –71 bp Glucose [cAMP] Inactive CAP Active CAP cAMPcAMP A T T A A T N N G C T A G C A T N N T A N N N N N N N N T A C G A T N N A T T A T A Consensus % Occurrence FIGURE 29.14 The mechanism of catabolite repression and CAP action. Glucose instigates catabolite repression by lowering cAMP levels. cAMP is necessary for CAP binding near promoters of operons whose gene prod- ucts are involved in the metabolism of alternative en- ergy sources such as lactose, galactose, and arabinose. The binding sites for the CAP–(cAMP) 2 complex are consensus DNA sequences containing the conserved pentamer TGTGA and a less well conserved inverted re- peat,TCANA (where N is any nucleotide). 918 Chapter 29 Transcription and the Regulation of Gene Expression Operons can also be classified as inducible, repressible, or both, depending on how they respond to the small molecules that mediate their expression. Repressible oper- ons are expressed only in the absence of their co-repressors. Inducible operons are transcribed only in the presence of small-molecule co-inducers (Figure 29.16). The araBAD Operon Is Both Positively and Negatively Controlled by AraC E. coli can use the plant pentose L-arabinose as sole source of carbon and energy. Ara- binose is metabolized via conversion to D-xylulose-5-P (a pentose phosphate pathway intermediate and transketolase substrate [see Chapter 22]) by three enzymes en- coded in the araBAD operon. Transcription of this operon is regulated by both catabolite repression and arabinose-mediated induction. CAP functions in catabolite repression; arabinose induction is achieved via the product of the araC gene, which FIGURE 29.15 Binding of CAP–(cAMP) 2 induces a severe bend in DNA about the center of dyad symmetry at the CAP-binding site.The CAP dimer with two molecules of cAMP bound interacts with 27 to 30 base pairs of du- plex DNA.Two ␣-helices of the CAP dimer insert into the major groove of the DNA at the dyad-symmetric CAP-binding site.The two cAMP molecules bound by the CAP dimer are indicated in yellow. Binding of CAP–(cAMP) 2 to its specific DNA site involves H bonding and ionic interactions between protein functional groups and DNA phosphates, as well as H-bonding in- teractions in the DNA major groove between amino acid side chains of CAP and DNA base pairs (pdb id ϭ 1CGP). Repressor Co-inducer Negative control Positive control InductionRepression Inactive repressor DNA mRNA Lactose operon Repressor deletions are constitutive Inactive inducer Co-inducer Active inducer DNA mRNA Catabolite repression Inducer deletions are uninducible DNA mRNA Tryptophan operon Repressor deletions are constitutive (de-repressed) Active inducer Corepressor Inactive inducer DNA mRNA Inactive repressor Corepressor Active repressor Inducer deletions are uninducible FIGURE 29.16 Control circuits gov- erning the expression of genes. These circuits can be either negative or positive, inducible or repressible. 29.2 How Is Transcription Regulated in Prokaryotes? 919 lies next to the araBAD operon on the E. coli chromosome. The araC gene product, the protein AraC, 4 is a 292-residue protein consisting of an N-terminal domain (residues 1 to 170) that binds arabinose and acts as a dimerization motif and a C-terminal (residues 178 to 292) DNA-binding domain. Regulation of araBAD by AraC is novel in that it acts both negatively and positively. The ara operon has three binding sites for AraC: araO 1 , located at nucleotides Ϫ106 to Ϫ144 relative to the araBAD transcription start site; araO 2 (spanning positions Ϫ265 to Ϫ294); and araI, the araBAD promoter. The araI site consists of two “half-sites”; araI 1 (nucleotides Ϫ56 to Ϫ78) and araI 2 (Ϫ35 to Ϫ51). (The araO 1 site contributes minimally to ara operon regulation.) The details of araBAD regulation are as follows: When AraC protein levels are low, the araC gene is transcribed from its promoter p c (adjacent to araO 1 ) by RNA poly- merase (Figure 29.17). araC is transcribed in the direction away from araBAD. When cAMP levels are low and arabinose is absent, an AraC protein dimer binds to two sites, araO 2 and the araI 1 half-site, forming a DNA loop between them and restricting tran- scription of araBAD (Figure 29.17). In the presence of L-arabinose, the monomer of AraC bound to the araO 2 site is released from that site; it then associates with the un- occupied araI half-site, araI 2 . L-Arabinose thus behaves as an allosteric effector that al- ters the conformation of AraC. In the arabinose-liganded conformation, the AraC dimer interacts with CAP–(cAMP) 2 to activate transcription by RNA polymerase. Thus, AraC protein is both a repressor and an activator. 4 Proteins are often named for the genes encoding them. By convention, the name of the protein is capitalized but not italicized. (a) The araBAD operon (b) Low [cAMP], no L-arabinose (c) High [cAMP], L-arabinose present DNA araI 2 araA araD RNA pol site araC araBaraI 1 araCaraO 2 CAP site araO 1 p C Regulatory gene D-Xylulose-5-PL-Ribulose-5-PL-RibuloseL-Arabinose L-Arabinose isomerase L-Ribulokinase Ribulose-5-P epimerase araI 2 araI 1 CAP site araA araD RNA polymerase araB araO 2 araO 1 araBAD mRNA araC araC Structural genes L-Arabinose araC araC araI 2 araA araD RNA pol site araBaraI 1 araC araO 2 CAP site araO 1 araC cAMP cAMP CAP CAP ADP ATP FIGURE 29.17 Regulation of the araBAD operon by the combined action of CAP and AraC protein. 920 Chapter 29 Transcription and the Regulation of Gene Expression Positive control of the araBAD operon occurs in the presence of L-arabinose and cAMP. Arabinose binding by AraC protein causes the release of araO 2 , opening of the DNA loop, and association of AraC with araI 2 . CAP–(cAMP) 2 binds at a site be- tween araO 1 and araI, and together the AraC–(arabinose) 2 and CAP–(cAMP) 2 com- plexes influence RNA polymerase through proteinϺprotein interactions to create an active transcription initiation complex. Supercoiling-induced DNA looping may promote proteinϺprotein interactions between DNA-binding proteins by bringing them into juxtaposition. The trp Operon Is Regulated Through a Co-Repressor–Mediated Negative Control Circuit The trp operon of E. coli (and S. typhimurium) encodes the five polypeptides, trpE through trpA (Figure 29.18), that assemble into the three enzymes catalyzing tryp- tophan synthesis from chorismate (see Chapter 25). Expression of the trp operon is under the control of Trp repressor, a dimer of 108-residue polypeptide chains. When tryptophan is plentiful, Trp repressor binds two molecules of tryptophan and associates with the trp operator that is located within the trp promoter. Trp repres- sor binding excludes RNA polymerase from the promoter, preventing transcription of the trp operon. When Trp becomes limiting, repression is lifted because Trp re- pressor lacking bound Trp (Trp apo-repressor) has a lowered affinity for the trp pro- moter. Thus, the behavior of Trp repressor corresponds to a co-repressor–mediated, negative control circuit (see Figure 29.16). Trp repressor not only is encoded by the trpR operon but also regulates expression of the trpR operon. This is an example of autogenous regulation (autoregulation): regulation of gene expression by the prod- uct of the gene. Attenuation Is a Prokaryotic Mechanism for Post-Transcriptional Regulation of Gene Expression In addition to repression, expression of the trp operon is controlled by transcription attenuation. Unlike the mechanisms discussed thus far, attenuation regulates tran- scription after it has begun. Charles Yanofsky, the discoverer of this phenomenon, has DNA trpB trpA L-TryptophanIndole-3-glycerol-PEnol-1-o-carboxy- phenylamino-1- deoxyribulose phosphate N-(5Ј-Phospho- ribosyl)- anthranilate Anthranilate synthase component I N-(5 Ј-Phosphoribosyl)- anthranilate isomerase Indole-3-glycerol phosphate synthase trpCtrpDtrpEtrpL Control sites trp p,O mRNA Anthranilate synthase component II Tryptophan synthase ␤-subunit Tryptophan synthase ␣-subunit Tryptophan synthase (␣ 2 ␤ 2 ) Anthranilate synthase (CoI 2 CoII 2 ) L-Serine Glyceraldehyde-3-P Anthranilate P P PRPP Chorismate Glutamine Glutamate + pyruvate CO 2 Attenuator FIGURE 29.18 The trp operon of E. coli. 29.2 How Is Transcription Regulated in Prokaryotes? 921 defined attenuation as any regulatory mechanism that manipulates transcription termination or transcription pausing to regulate gene transcription downstream. In prokaryotes, tran- scription and translation (see Chapters 10 and 30) are coupled, and the translating ribosome is affected by the formation and persistence of secondary structures in the mRNA. In many operons encoding enzymes of amino acid biosynthesis, a transcribed 150- to 300-bp leader region is positioned between the promoter and the first major structural gene. These regions encode a short leader peptide containing multiple codons for the pertinent amino acid. For example, the leader peptide of the leu operon has four leucine codons, the trp operon has two tandem tryptophan codons, and so forth (Figure 29.19). Translation of these codons depends on an adequate sup- ply of the relevant aminoacyl-tRNA, which in turn rests on the availability of the amino acid. When tryptophan is scarce, the entire trp operon from trpL to trpA is tran- scribed to give a polycistronic mRNA. But, as [Trp] increases, more and more of the trp transcripts consist of only a 140-nucleotide fragment corresponding to the 5Ј-end of trpL. Tryptophan availability is causing premature termination of trp transcription, that is, transcription attenuation. Although attenuation occurs when tryptophan is abundant, attenuation is blocked when levels of tryptophan are low and little trypto- phanyl-tRNA is available. The secondary structure of the 160-bp leader region tran- script is the principal control element in transcription attenuation (Figure 29.20). This RNA segment includes the coding region for the 14-residue leader peptide. Three critical base-paired hairpins can form in this RNA: the 1 Ϻ 2 pause structure, the Methis Thr Arg Val Gln Phe Lys His His His His His His His Pro Asp Met ilv Thr Ala Leu Leu Arg Val Ile Ser Leu Val Val Ile Ser Val Val Val Ile Ile Ile Pro Pro Cys Gly Ala Ala Leu Gly Arg Gly Lys Ala Met leu Ser His Ile Val Arg Phe Thr Gly Leu Leu Leu Leu Asn Ala Phe Ile Val Arg Gly Arg Pro Val Gly Gly Ile Gln His Met p heA Lys His Ile Pro Phe Phe Phe Ala Phe Phe Phe Thr Phe Pro Met thr Lys Arg Ile Ser Thr Thr Ile Thr Thr Thr Ile Thr Ile Thr Thr Gln Asn Gly Ala Gly Met trp Lys Ala Ile Phe Val Leu Lys Gly Trp Trp Arg Thr Ser Operon Amino acid Sequence FIGURE 29.19 Amino acid sequences of leader peptides in various amino acid biosynthetic operons regulated by attenuation. Color indicates amino acids synthesized in the pathway catalyzed by the operon’s gene prod- ucts. (The ilv operon encodes enzymes of isoleucine, leucine, and valine biosynthesis.) A50 G G U UA GC GC UA GC G C G CG AU CG UA U CG CG A A C G C U A U U GC UA CG G U A A A A U C A G A U A C C C AU GC CG CG CG GC CG UUUUUU C U A A U G A Trp Trp 70 110 130 A G G U U G G U G G C G C A C U U C C A A CG GC GC GC CG A G U A C C GC UA AU U UA CG A C C A UA GC CG G U A A A A C U A C C U A A U G A G U C G G G C UUUU 130 UU 50 110 70 Trp codons Stop codon for leader peptide 1 • 2 3 •4 “Terminator” 2 •3 “Antiterminator” Pause Structure 92 92 U G A U G A FIGURE 29.20 Alternative secondary struc- tures for the leader region (trpL mRNA) of the trp operon transcript. 922 Chapter 29 Transcription and the Regulation of Gene Expression 3 Ϻ 4 terminator, and the 2 Ϻ 3 antiterminator. Obviously, the 1Ϻ2 pause, 3Ϻ4 terminator, and the 2Ϻ3 antiterminator represent mutually exclusive alternatives. A significant fea- ture of this coding region is the tandem UGG tryptophan codons. Transcription of the trp operon by RNA polymerase beg ins and progresses un- til position 92 is reached, whereupon the 1Ϻ2 hairpin is formed, causing RNA polymerase to pause in its elongation cycle. While RNA polymerase is paused, a ribosome begins to translate the leader region of the transcript. Translation by the ribosome releases the paused RNA polymerase and transcription continues, with RNA polymerase and the ribosome moving in unison. As long as tryptophan is plentiful enough that tryptophanyl-tRNA Tr p is not limiting, the ribosome is not delayed at the two tryptophan codons and follows closely behind RNA poly- merase, translating the message soon after it is transcribed. The presence of the ribosome atop segment 2 blocks formation of the 2Ϻ3 antiterminator hairpin, al- lowing the alternative 3Ϻ4 terminator hairpin to form (Figure 29.21). Stable hair- pin structures followed by a run of Us are features typical of rho-independent transcription termination signals, so the RNA polymerase perceives this hairpin as a transcription stop signal and transcription is terminated at this point. On the other hand, a paucity of tryptophan and hence low availability of tryptophanyl- tRNA Tr p causes the ribosome to stall on segment 1. This leaves segment 2 free to pair with segment 3 and to form the 2Ϻ3 antiterminator hairpin in the transcript. Because this hairpin precludes formation of the 3Ϻ4 terminator, termination is prevented and the entire operon is transcribed. Thus, transcription attenuation is determined by the availability of tyrptophanyl-tRNA Tr p and its transitory influ- ence over the formation of alternative secondary structures in the mRNA. DNA Ϻ Protein Interactions and Protein Ϻ Protein Interactions Are Essential to Transcription Regulation Quite a variety of control mechanisms regulate transcription in prokaryotes. Several organizing principles materialize. First, DNA Ϻ protein interactions are a central fea- ture in transcriptional control, and the DNA sites where regulatory proteins bind commonly display at least partial dyad symmetry or inverted repeats. Furthermore, DNA-binding proteins themselves are generally even-numbered oligomers (for ex- ample, dimers, tetramers) that have an innate twofold rotational symmetry. Second, Antiterminator trp operon mRNA Transcribing RNA polymerase “Terminated” RNA polymerase Ribosome stalled at tandem trp codons (b) Low tryptophan High tryptophan(a) trpL mRNA DNA encoding trp operon Transcription terminator Ribosome transcribing the leader peptide RNA + FIGURE 29.21 The mechanism of attenuation in the trp operon.