Because the structure and function of a cell are determined by the proteins it contains, the control of gene expression is a fundamental aspect of molecular cell biology. Most commonly, the “decision” to transcribe the gene encoding a particular protein is the major mechanism for controlling production of the encoded protein in a cell. By controlling transcription, a cell can regulate which proteins it produces and how rapidly they are synthesized. When transcription of a gene is repressed, the corresponding mRNA and encoded protein or proteins are synthesized at low rates. Conversely, when transcription of a gene is activated, both the mRNA and encoded protein or proteins are produced at much higher rates.

In most bacteria and other single-celled organisms, gene expression is highly regulated in order to adjust the cell’s enzymatic machinery and structural components to changes in the nutritional and physical environment. Thus at any given time, a bacterial cell normally synthesizes only those proteins that are required for its survival under the current conditions. Here we describe the basic features of transcriptional control in bacteria, using the lac operon and the glutamine synthetase gene in E. coli and the xpt-pbuX operon in Bacillus subtilis as our primary examples. Many of the same features are involved in eukaryotic transcriptional control, which will be the subject of the remainder of this chapter.

Transcription Initiation by Bacterial RNA Polymerase Requires Association with a Sigma Factor

In E. coli, about half the genes are clustered into operons, each of which encodes enzymes involved in a particular metabolic pathway or proteins that interact to form one multisubunit protein complex. For instance, the trp operon discussed in Chapter 5 encodes five polypeptides needed in the biosynthesis of tryptophan. Similarly, the lac operon encodes three proteins required for the metabolism of lactose, a sugar present in milk. Because a bacterial operon is transcribed from one start site into a single mRNA, all the genes within an operon are coordinately regulated; that is, they are all activated or repressed at the same time to the same extent.

The transcription of operons, as well as that of isolated genes, is controlled by interplay between RNA polymerase and specific repressor and activator proteins. In order to initiate transcription, E. coli RNA polymerase must associate with one of a small number of σ (sigma) factors. The most common one in eubacterial cells is σ70. This σ-factor binds to both RNA polymerase and promoter DNA sequences, bringing the RNA polymerase enzyme to the promoter. It recognizes and binds to both a six-base-pair sequence centered at about 10 bp and a seven-base-pair sequence centered at about 35 bp upstream from the +1 transcription start. Consequently, the −10 sequence and the −35 sequence together constitute a promoter for E. coli RNA polymerase associated with σ70. Although the promoter sequences contacted by σ70 are located at −35 and −10, E. coli RNA polymerase binds to the promoter-region DNA from roughly −50 to +20 through interactions with DNA that do not depend on the sequence. The σ-factor also assists the RNA polymerase in separating the DNA strands at the transcription start site and in inserting the coding strand into the active site of the polymerase so that transcription starts at +1. The optimal σ70-RNA polymerase promoter sequence, determined as the “consensus sequence” of multiple strong promoters, is

This consensus sequence shows the most commonly occur ring base at each of the positions in the −35 and −10 regions. The size of the font indicates the importance of the base at that position, as determined by the influence of mutations of these bases on the frequency of transcription initiation (i.e., the number of times per minute that RNA polymerases initiate transcription). The sequence shows the strand of DNA that has the same 5′→3′ orientation as the transcribed RNA (i.e., the nontemplate strand). However, the σ70-RNA polymerase initially binds to double-stranded DNA. After the polymerase transcribes a few tens of base pairs, σ70 is released. Thus σ70 acts as an initiation factor that is required for transcription initiation, but not for RNA strand elongation once initiation has taken place.

Initiation of lac Operon Transcription Can Be Repressed or Activated

When E. coli is in an environment that lacks lactose, syn thesis of lac mRNA is repressed so that cellular energy is not wasted synthesizing enzymes the cell does not require. In an environment containing both lactose and glucose, E. coli cells preferentially metabolize glucose, the central molecule of carbohydrate metabolism. The cells metabolize lactose at a high rate only when lactose is present and glucose is largely depleted from the medium. They achieve this metabolic adjustment by repressing transcription of the lac operon until lactose is present and allowing synthesis of only low levels of lac mRNA until the cytosolic concentration of glucose falls to low levels. Transcription of the lac operon under different conditions is controlled by lac repressor protein and catabolite activator protein (CAP) (also called CRP, for cAMP receptor protein), each of which binds to a specific DNA sequence in the lac transcription-control region; these two sequences are called the operator and the CAP site, respectively (Figure 1, top).

Fig1. Regulation of transcription from the lac operon of E. coli. (Top) The transcription-control region, composed of roughly a hundred base pairs, includes three protein-binding regions: the CAP site, which binds catabolite activator protein; the lac promoter, which binds the σ70-RNA polymerase complex; and the lac opera tor, which binds lac repressor. The lacZ gene encoding the enzyme β-galactosidase, the first of the three genes in the operon, is shown to the right. (a) In the absence of lactose, very little lac mRNA is produced because the lac repressor binds to the operator, inhibiting transcription initiation by σ70-RNA polymerase. (b) In the presence of glucose and lactose, lac repressor binds lactose and dissociates from the operator, allowing σ70-RNA polymerase to initiate transcription at a low rate. (c) Maximal transcription of the lac operon occurs in the presence of lactose and the absence of glucose. In this situation, cAMP increases in response to the low glucose concentration and forms a CAP-cAMP complex, which binds to the CAP site, where it interacts with RNA polymerase to increase the rate of transcription initiation. (d) The tetrameric lac repressor binds to the primary lac operator (O1) and one of two secondary operators (O2 or O3) simultaneously. The two structures are in equilibrium. See B. Muller-Hill, 1998, Curr. Opin. Microbiol. 1:145. [Part (d) data from M. Lewis et al., 1996, Science 271:1247-1254, PDB IDs 1lbh and 1lbg; and R. Daber et al., 2007, J. Mol. Biol. 370:609-619, PDB ID 2pe5.]

For transcription of the lac operon to begin, the σ70 sub unit of the RNA polymerase must bind to the lac promoter at the −35 and −10 promoter sequences. When no lactose is present, the lac repressor binds to the lac operator, which overlaps the transcription start site. Therefore, the lac re pressor bound to the operator site blocks σ70 binding and hence transcription initiation by RNA polymerase (Figure 1a). When lactose is present, it binds to specific binding sites in each subunit of the tetrameric lac repressor, causing a conformational change in the protein that makes it dis sociate from the lac operator. As a result, the polymerase can bind to the promoter and initiate transcription of the lac operon. However, when glucose is also present, the frequency of transcription initiation is very low, resulting in the synthesis of only low levels of lac mRNA and thus of the proteins encoded by the lac operon (Figure 1b). The frequency of transcription initiation is low because the −35 and −10 sequences in the lac promoter differ from the ideal σ70-binding sequences shown previously.

Once glucose is depleted from the medium and the intracellular glucose concentration falls, E. coli cells respond by synthesizing cyclic AMP (cAMP). As the concentration of cAMP increases, it binds to a site in each subunit of the dimeric CAP protein, causing a conformational change that allows the protein to bind to the CAP site in the lac transcription-control region. The bound CAP-cAMP complex interacts with the polymerase bound to the promoter, greatly increasing the frequency of transcription initiation. This activation leads to synthesis of high levels of lac mRNA and subsequently of the enzymes encoded by the lac operon (Figure1c).

In fact, the lac operon is more complex than depicted in the simplified model in Figure 1a–c. The tetrameric lac repressor actually binds to two DNA sequences simultaneously, one at the primary operator (lacO1), which over laps the region of DNA bound by RNA polymerase at the promoter, and the other at one of two secondary operators centered at +412 (lacO2), within the lacZ protein-coding region, and −82 (lacO3) (Figure 1d). The lac repressor tetramer is a dimer of dimers. Each dimer binds to one operator (Figure 1d). Simultaneous binding of the tetrameric lac repressor to the primary lac operator and one of the two secondary operators is possible because DNA is quite flexible, as we saw in the wrapping of DNA around the surface of a histone octamer in the nucleosomes of eukaryotes. The secondary operators function to increase the local concentration of lac repressor in the micro-vicinity of the primary operator where repressor binding blocks RNA polymerase binding. Since the equilibrium of binding reactions depends on the concentrations of the binding partners, the resulting increased local concentration of lac repressor in the vicinity of O1 increases repressor binding to O1. There are approximately 10 lac repressor tetramers per E. coli cell. Because of binding to O2 and O3, there is nearly always a lac repressor tetramer much closer to O1 than would otherwise be the case if the 10 repressor tetramers were diffusing randomly through the cell. If both O2 and O3 are mutated so that the lac repressor no longer binds to them with high affinity, repression at the lac promoter is reduced by a factor of 70. Mutation of only O2 or only O3 reduces repression twofold, indicating that either one of these secondary opera tors can provide most of the increase in repression.

Although the promoters for different E. coli genes exhibit considerable homology, their exact sequences differ. The promoter sequence determines the intrinsic frequency at which RNA polymerase–σ complexes initiate transcription of a gene in the absence of a repressor or activator protein. Promoters that support a high frequency of transcription initiation have −10 and −35 sequences similar to the ideal promoter shown previously and are called strong promoters. Those that support a low frequency of transcription initiation differ from this ideal sequence and are called weak promoters. The lac operon, for instance, has a weak promoter whose sequence differs from the consensus strong promoter at several positions. Its low intrinsic frequency of initiation is further reduced by the lac repressor and substantially increased by the cAMP-CAP complex.

Small Molecules Regulate Expression of Many Bacterial Genes via DNA-Binding Repressors and Activators

Transcription of most E. coli genes is regulated by processes similar to those described for the lac operon, although the detailed interactions differ at each promoter. The general mechanism involves a specific repressor that binds to the operator region of a gene or operon, thereby blocking transcription initiation. A small-molecule ligand binds to the repressor controlling its DNA-binding activity, and consequently the frequency of transcription initiation and therefore the rate of synthesis of the mRNA and encoded proteins as appropriate for the needs of the cell. As for the lac operon, many eubacterial transcription-control regions contain one or more secondary operators that contribute to the level of repression.

Specific activator proteins, such as CAP in the lac operon, also control transcription of a subset of bacterial genes that have binding sites for the activator. Like CAP, other activators bind to DNA together with RNA polymerase, stimulating transcription from a specific promoter. The DNA-binding activity of an activator can be modulated in response to cellular needs by the binding of specific small molecule ligands (e.g., cAMP) or by post-translational modifications, such as phosphorylation, that alter the con formation of the activator.

Transcription Initiation from Some Promoters Requires Alternative Sigma Factors

Most E. coli promoters interact with σ70-RNA polymerase, the major initiating form of the bacterial enzyme. The transcription of certain groups of genes, however, is initiated by E. coli RNA polymerases containing one of several alternative sigma factors that recognize different consensus promoter sequences than σ70 does (Table1). These alternative σ-factors are required for the transcription of sets of genes with related functions, such as those involved in the response to heat shock or nutrient deprivation, motility, or sporulation in gram-positive eubacteria. In E. coli, there are 6 alternative σ-factors in addition to the major “house keeping” σ-factor, σ70. The genome of the gram-positive, sporulating bacterium Streptomyces coelicolor encodes 63 σ-factors, the current record, based on sequence analysis of hundreds of eubacterial genomes. Most are structurally and functionally related to σ70. Transcription initiation by RNA polymerases containing σ70-like factors is regulated by re pressors and activators that bind to DNA near the region where the polymerase binds. But one class, represented in E. coli by σ54, is unrelated to σ70 and functions differently.

Table1. Sigma Factors of E. coli

Transcription by σ54-RNA Polymerase Is Controlled by Activators That Bind Far from the Promoter

The sequence of σ54 is distinctly different from that of all the σ70-like factors. Transcription of genes by RNA polymerases containing σ54 is regulated solely by activators whose binding sites in DNA, referred to as enhancers, are generally located 80–160 bp upstream from the transcription start site. Even when enhancers are moved more than a kilobase away from a start site, σ54-activators can activate transcription.

The best-characterized σ54-activator—the NtrC protein (nitrogen regulatory protein C)—stimulates transcription of the glnA gene. The glnA gene encodes the enzyme glutamine synthetase, which synthesizes the amino acid glutamine, the central molecule of nitrogen metabolism, from glutamic acid and ammonia. The σ54-RNA polymerase binds to the glnA promoter but does not melt the DNA strands and initiate transcription until it is activated by NtrC, a dimeric protein. NtrC, in turn, is regulated by a protein kinase called NtrB. In response to low levels of glutamine, NtrB phosphorylates dimeric NtrC, which then binds to an enhancer upstream of the glnA promoter. Enhancer-bound phosphorylated NtrC then stimulates the σ54-polymerase bound at the promoter to separate the DNA strands and initiate transcription.

Electron microscopy studies have shown that phosphorylated NtrC bound at enhancers and σ54-polymerase bound at the promoter interact directly, forming a loop in the DNA between the binding sites (Figure 2). this activation mechanism resembles the predominant mechanism of transcriptional activation in eukaryotes.

Fig2. DNA looping permits interaction of bound NtrC and σ54-RNA polymerase. (a) Drawing (left) and electron micrograph (right) of DNA restriction fragment with phosphorylated NtrC dimers bound to the enhancer region near one end and σ54-RNA polymerase bound to the glnA promoter near the other end. (b) Drawing (left) and electron micrograph (right) of the same fragment preparation, showing NtrC dimers and σ54-RNA polymerase bound to each other, with the intervening DNA forming a loop between them. See W. Su et al., 1990, Proc. Natl. Acad. Sci. USA 87:5504. [Micrographs courtesy Harrison Echols and Carol Gross.]

NtrC has ATPase activity, and ATP hydrolysis is re quired for activation of bound σ54-RNA polymerase by phosphorylated NtrC. Mutants with an NtrC that is defective in ATP hydrolysis are invariably defective in stimulating the σ54-RNA polymerase to melt the DNA strands at the transcription start site. It is postulated that ATP hydrolysis supplies the energy required for melting the DNA strands. In contrast, the σ70-polymerase does not require ATP hydrolysis to separate the strands at a start site.

Many Bacterial Responses Are Controlled by Two-Component Regulatory Systems

As we have just seen, control of the E. coli glnA gene depends on two proteins, NtrC and NtrB. Such two- component regulatory systems control many responses of bacteria to changes in their environment. At high concentrations of glutamine, glutamine binds to a sensor domain of NtrB, causing a conformational change in the protein that inhibits its histidine kinase activity (Figure 3a). At the same time, the regulatory domain of NtrC blocks its DNA-binding domain from binding the glnA enhancers. At low concentrations of glutamine, glutamine dissociates from the sensor domain in the NtrB protein, leading to activation of a histidine kinase transmitter domain in NtrB that transfers the γ-phosphate of ATP to a histidine residue (H) in the transmitter domain. This phosphohistidine then transfers the phosphate to an aspartic acid residue (D) in the NtrC protein. This causes a conformational change in NtrC that unmasks the NtrC DNA-binding domain so that it can bind to the glnA enhancers.

Fig3. Two-component regulatory systems. (a) At low cytoplasmic concentrations of glutamine, glutamine dissociates from NtrB, resulting in a conformational change that activates a protein kinase transmitter domain that transfers an ATP γ-phosphate to a conserved histidine (H) in the transmitter domain. This phosphate is then transferred to an aspartic acid (D) in the regulatory domain of the response regulator NtrC. This converts NtrC into its activated form, which binds the enhancer sites upstream of the glnA promoter (see Figure 2). (b) General organization of two-component histidyl-aspartyl phospho-relay regulatory systems in bacteria and plants. See A. H. West and A. M. Stock, 2001, Trends Biochem. Sci. 26:369.

Many other bacterial responses are regulated by two proteins with homology to NtrB and NtrC (Figure 3b). In each of these regulatory systems, one protein, called a histidine kinase sensor, contains a latent histidine kinase transmitter domain that is regulated in response to environmental changes detected by a sensor domain. When activated, the transmitter domain transfers the γ-phosphate of ATP to a histidine residue in the transmitter domain. The second protein, called a response regulator, contains a receiver domain homologous to the region of NtrC containing the aspartic acid residue that is phosphorylated by activated NtrB. The response regulator contains a second functional domain that is regulated by phosphorylation of the receiver domain. In many cases, this domain of the response regulator is a sequence-specific DNA-binding domain that binds to related DNA sequences and functions either as a repressor, like the lac repressor, or as an activator, like CAP or NtrC, regulating the transcription of specific genes. However, the effector domain can have other functions as well, such as controlling the direction in which the bacterium swims in response to a concentration gradient of nutrients. Although all transmitter domains are homologous (as are receiver domains), the transmitter domain of a specific sensor protein will phosphorylate only the receiver domains of specific response regulators, allowing specific responses to different environmental changes. Similar two-component histidyl-aspartyl phospho-relay regulatory systems are also found in plants.

Expression of Many Bacterial Operons Is Controlled by Regulation of Transcriptional Elongation

 In addition to regulation of transcription initiation by activators and repressors, expression of many bacterial operons is controlled by regulation of transcriptional elongation in the promoter-proximal region. This mechanism of control was first discovered in studies of trp operon transcription in E. coli. Transcription of the trp operon is repressed by the trp repressor when the concentration of tryptophan in the cytoplasm is high. But the low level of transcription initiation that still occurs is further controlled by a process called attenuation when the concentration of charged tRNA^Trp is sufficient to support a high rate of protein synthesis. The first 140 nt of the trp operon does not encode proteins required for tryptophan biosynthesis, but rather consists of a short peptide “leader sequence,” as diagrammed in Figure 4a. Region 1 of this leader sequence contains two successive Trp codons. Region 3 can base-pair with either region 2 or region 4. A ribosome follows closely behind the RNA polymerase, initiating translation of the leader peptide shortly after the 5′ end of the trp leader sequence emerges from the RNA polymerase. When the concentration of tRNA^Trp is sufficient to support a high rate of protein synthesis, the ribosome translates quickly through region 1 into region 2, blocking the ability of region 2 to base-pair with region 3 as it emerges from the surface of the transcribing RNA polymerase (Figure 4b, left). Instead, region 3 base-pairs with region 4 as soon as it emerges from the surface of the polymerase, forming a stem-loop followed by several uracils, which is a signal for bacterial RNA polymerase to pause transcription and terminate. As a consequence, the remainder of the long trp operon is not transcribed, and the cell does not waste the energy required for tryptophan synthesis, or for the translation of the encoded proteins, when the concentration of tryptophan is high.

Fig4. Transcriptional control by regulation of RNA polymerase elongation and termination in the E. coli trp operon. (a) Diagram of the 140-nucleotide trp leader RNA. The numbered regions are critical to attenuation. (b) Translation of the trp leader sequence begins near the 5′ end soon after it is transcribed, while transcription of the rest of the polycistronic trp mRNA molecule continues. At high concentrations of charged tRNA^Trp, formation of the 3–4 stem loop followed by a series of uracils causes termination of transcription. At low concentrations of charged tRNA^Trp, region 3 is sequestered in the 2–3 stem-loop and cannot base-pair with region 4. In the absence of the stem-loop structure required for termination, transcription of the trp operon continues. See C. Yanofsky, 1981, Nature 289:751.

However, when the concentration of tRNA^Trp is not sufficient to support a high rate of protein synthesis, the ribosome stalls at the two successive Trp codons in region 1 (Figure 4b, right). As a consequence, region 2 base-pairs with region 3 as soon as it emerges from the transcribing RNA polymerase. This prevents region 3 from base-pairing with region 4, so the 3–4 hairpin does not form and does not cause RNA polymerase pausing or transcription termination. As a result, the proteins required for tryptophan syn thesis are translated by ribosomes that initiate translation at the start codons for each of these proteins in the long polycistronic trp mRNA.

Attenuation of transcription elongation also occurs at some operons and single genes encoding enzymes involved in the biosynthesis of other amino acids and metabolites through the function of riboswitches. Riboswitches are sequences of RNA most commonly found in the 5′ untranslated region of bacterial mRNAs. They fold into complex tertiary structures called aptamers that bind small-molecule metabolites when those metabolites are present at sufficiently high concentrations. In some cases, this binding results in the formation of stem-loop structures that lead to early termination of transcription, as in the Bacillus subtilis xpt-pbuX operon, which encodes enzymes involved in purine synthesis (Figure 5). When the concentration of small-molecule metabolites is lower, the metabolites are not bound by the aptamers, and alternative RNA structures form that do not induce transcription termination, allowing transcription of genes encoding enzymes involved in the syn thesis of the metabolites. As we will see below, although the mechanism in eukaryotes is different, regulation of promoter proximal transcriptional pausing and termination has recently been discovered to occur frequently in the regulation of gene expression in multicellular organisms as well.

Fig5. Riboswitch control of transcription termination in B. subtilis. (a) During transcription of the Bacillus subtilis xpt-pbuX operon, which encodes enzymes involved in purine synthesis, the 5′ untranslated region of the mRNA can fold into alternative structures depending on the concentration of purines in the cytoplasm, forming the “purine riboswitch.” At high concentrations of purines, the riboswitch folds into an aptamer that binds a purine ligand (cyan circle), allowing formation of a stem-loop transcription termination signal similar to the termination signal that forms in the E. coli trp operon mRNA at high tryptophan concentrations (see Figure 4), i.e., a stem loop followed by a run of Us. At low purine concentrations, an alternative RNA structure forms that prevents formation of the transcription termination signal, permitting transcription of the operon. Note the alternative base pairing of the red and blue regions of the RNA. (b) Structure of the purine riboswitch bound to a purine (cyan) as determined by X-ray crystallography. See A. D. Garst, A. L. Edwards, and R. T. Batey, 2011, Cold Spring Harb. Perspect. Biol. 3:a003533. [Part (b) data from R. T. Batey, S. D. Gilbert, and R. K. Montagne, 2004, Nature 432:411, PDB ID 4fe5.]

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