Transcriptional Control of Gene Expression PDF

CHAPTER 9 Transcriptional...

CHAPTER 9 Transcriptional Control of Gene Drosophila polytene chromosomes stained with antibodies against a Expression chromatin-remodeling ATPase called Kismet (blue), RNA polymerase II with low CTD phosphorylation (red), and RNA polymerase II with high CTD phos- phorylation (green). [Reproduced with permission of The Company of Biologists, from Srinivasan, S., et al., “The Drosophila trithorax group protein Kismet facilitates an early step in transcriptional elongation by RNA Polymerase II,” Development, 2005, 132(7):1623-1635; permission conveyed through Copyright Clearance Center, Inc.] In previous chapters, we have seen that the properties and also plays a vital role in bacteria and other single-celled functions of each cell type are determined by the proteins it microorganisms, in which it allows cells to adjust their enzy- contains. In this chapter and the next, we consider how the matic machinery and structural components in response to kinds and amounts of the various proteins produced by a their changing nutritional and physical environment. Conse- particular cell type in a multicellular organism are regulated. quently, to understand how microorganisms respond to their This regulation of gene expression is the fundamental pro- environment and how multicellular organisms normally cess that controls the development of multicellular organ- develop, as well as how pathological abnormalities of gene isms such as ourselves from a single fertilized egg cell into expression occur, it is essential to understand the molecular the thousands of cell types of which we are made. When interactions that control protein production. gene expression goes awry, cellular properties are altered, a The basic steps in gene expression—that is, the entire pro- process that all too often leads to the development of cancer. cess whereby the information encoded in a particular gene is As discussed further in Chapter 24, genes encoding proteins decoded into a particular protein—are reviewed in Chapter 5. that restrain cell growth are abnormally repressed in can- Synthesis of mRNA requires that an RNA polymerase initiate cer cells, whereas genes encoding proteins that promote cell transcription (initiation), polymerize ribonucleoside triphos- growth and replication are inappropriately activated in can- phates complementary to the DNA coding strand (elongation), cer cells. Abnormalities in gene expression also result in de- and then terminate transcription (termination) (see Figure velopmental defects such as cleft palate, tetralogy of Fallot (a 5-11). In bacteria, ribosomes and translation initiation fac- serious developmental defect of the heart that can be treated tors have immediate access to newly formed RNA transcripts, surgically), and many others. Regulation of gene expression which function as mRNA without further modification. OUTLINE 9.1 Control of Gene Expression in Bacteria 9.5 Molecular Mechanisms of Transcription Repression and Activation 9.2 Overview of Eukaryotic Gene Control 9.6 Regulation of Transcription-Factor Activity 9.3 RNA Polymerase II Promoters and General Transcription Factors 9.7 Epigenetic Regulation of Transcription 9.4 Regulatory Sequences in Protein-Coding Genes and 9.8 Other Eukaryotic Transcription Systems the Proteins Through Which They Function In eukaryotes, however, the initial RNA transcript is sub- alter the structures of flowers in plants (Figure 9-2c), and are jected to processing that yields a functional mRNA (see responsible for multiple other developmental abnormalities. Figure 5-15). The mRNA then is transported from its site of Transcription is a complex process involving many layers synthesis in the nucleus to the cytoplasm, where it is trans- of regulation. In this chapter, we focus on the molecular events lated into protein with the aid of ribosomes, tRNAs, and that determine when transcription of a gene occurs. First, translation factors (see Figures 5-23, 5-24, and 5-26). we consider the mechanisms of gene expression in bacteria, Regulation may occur at several of the various steps in gene in which DNA is not bound by histones and packaged into expression outlined above: transcription initiation, elongation, nucleosomes. Repressor and activator proteins recognize and RNA processing, and mRNA export from the nucleus, as well bind to specific DNA sequences to control the transcription of as through control of mRNA degradation, mRNA translation a nearby gene, and in many cases, specific tertiary structures in into protein, and protein degradation. This regulation results in nascent mRNAs, called riboswitches, bind metabolites to reg- differential protein expression in different cell types or develop- ulate transcription elongation. The remainder of the chapter mental stages or in response to external conditions. Although focuses on eukaryotic regulation of transcription and how the examples of regulation at each step in gene expression have been basic tenets of bacterial regulation are applied in more com- found, control of transcription initiation and of elongation— plex ways in higher organisms. In addition, eukaryotic regula- the first two steps—are the most important mechanisms for de- tion mechanisms make use of the association of DNA with termining whether most genes are expressed and how much of histone octamers, forming chromatin structures with varying the encoded mRNAs and, consequently, proteins are produced degrees of condensation, and of post-translational modifica- (Figure 9-1). The molecular mechanisms that regulate transcrip- tions of histone tails such as acetylation and methylation (see tion initiation and elongation are critical to numerous biologi- Figure 8-26). Figure 9-3 provides an overview of transcrip- cal phenomena, including the development of a multicellular tional regulation in metazoans (multicellular animals) and organism, as mentioned above, the immune responses that of the processes outlined in this chapter. We discuss how the protect us from pathogenic microorganisms, and neurological RNA polymerases responsible for the transcription of different processes such as learning and memory. When these regula- classes of eukaryotic genes bind to promoter sequences to initi- tory mechanisms controlling transcription function improp- ate the synthesis of an RNA molecule, and how specific DNA erly, pathological processes may occur. For example, dominant sequences function as transcription-control regions by serving mutations of the HOXD13 gene result in polydactyly, the em- as the binding sites for the transcription factors that regulate bryological development of extra digits of the feet, hands, or transcription. Next we consider how eukaryotic activators and both (Figure 9-2a). HOXD13 encodes a transcription factor repressors influence transcription through interactions with that normally regulates the transcription of multiple genes in- large multiprotein complexes. Some of these multiprotein com- volved in development of the extremities. Other mutations af- plexes modify chromatin condensation, altering the accessibi- fecting the function or expression of transcription factors cause lity of chromosomal DNA to transcription factors and RNA an extra pair of wings to develop in Drosophila (Figure 9-2b), polymerases. Other complexes directly influence the frequency at which RNA polymerases bind to promoters and initiate transcription. Very recent research has revealed that, for many Rates of: genes in multicellular animals, the RNA polymerase pauses Transcription after transcribing a short RNA, and that one transcriptional regulation mechanism involves a release of the paused poly- 73% mRNA translation 8% merase, allowing it to transcribe the rest of the gene. We discuss 8% how transcription of specific genes can be specified by par- Protein degradation ticular combinations of the roughly 1400 transcription factors 11% encoded in the human genome, giving rise to cell-type-specific mRNA degradation gene expression. We consider the various ways in which the activities of transcription factors themselves are controlled to FIGURE 91 Contributions of the major processes that regulate ensure that genes are expressed only in the correct cell types protein concentrations. The concentration of a protein is controlled and at the appropriate time during their differentiation. by regulation of the frequency with which the mRNA encoding the pro- We also discuss recent studies revealing that RNA-protein tein is synthesized (gene transcription), the rate at which that mRNA is complexes in the nucleus can regulate transcription. New degraded, the rate at which that mRNA is translated into protein, and methods for sequencing DNA, coupled with reverse tran- the rate at which that protein is degraded. The relative contributions scription of RNA into DNA in vitro, have revealed that of these four rates to determining the concentrations of thousands of proteins in cultured mouse fibroblasts were determined by mass much of the genome of eukaryotes is transcribed into low- spectrometry to measure protein concentrations (see Chapter 3), abundance RNAs that do not encode proteins. Several nu- mRNA sequencing (RNA-seq) to measure mRNA levels (see Chapter 6), clear long noncoding RNAs (lncRNAs) have recently been protection of mRNA from ribonuclease digestion by associated discovered to regulate the transcription of other protein- ribosomes (ribosome footprinting) to estimate translation rates, stable coding genes. This finding raises the possibility that tran- isotope labeling to determine degradation rates, and statistical analysis scriptional control by such noncoding RNAs may be much of the data to correct for inherent biases and errors in these methods. more general than is currently understood. Recent advances [Data from J. J. Li and M. D. Biggin, 2014, Science 347:1066.] in mapping the association of transcription factors with 354 CHAPTER 9 t Transcriptional Control of Gene Expression specific regions of chromatin across the entire genome in a for controlling eukaryotic gene expression are covered in variety of cell types have provided the first glimpses of how Chapter 10. Subsequent chapters, particularly Chapters 15, transcription factors regulate embryonic development from 16, and 21, provide examples of how transcription is reg- the pluripotent stem cells of the early embryo to the fully ulated by interactions between cells and how the resulting differentiated cells that make up most of our tissues. RNA gene control contributes to the development and function of processing and various post-transcriptional mechanisms specific types of cells in multicellular organisms. (a) Normal Dominant HOXD13 mutation (b) Haltere Normal Ubx mutation (c) Homozygous recessive mutations in Normal ap2-1, pi-1, and ag-1 genes FIGURE 92 Phenotypes of mutations in genes encoding affect master regulatory transcription factors that regulate multiple transcription factors. (a) A dominant mutation in the human HOXD13 genes, including many genes encoding other transcription factors. gene results in the development of extra digits, a condition known as [Part (a), left, Lightvision, LLC/Moment Open/Getty Images; right, Goodman, polydactyly. (b) Homozygous recessive mutations that prevent expres- F. R. and Scrambler, P. J., Human HOX gene mutations. Clinical Genetics, 2001, sion of the Ubx gene in the third thoracic segment of Drosophila result 59:1, pages 1–11. Part (b) from “The bithorax complex: the first fifty years,” by in transformation of that segment, which normally has a balancing Edward B. Lewis, reproduced with permission from The International Journal of organ called a haltere, into a second copy of the thoracic segment that Developmental Biology, 1998, Vol 42(403-15), Figures 4a and 4b. Part (c) repub- develops wings. (c) Mutations in Arabidopsis thaliana that inactivate lished with permission of Elsevier, from Weigel, D. and Meyerowitz, M., “The both copies of three floral organ–identity genes transform the normal ABCs of floral homeotic genes,” Cell, 1994, 78(2):203-209; permission conveyed parts of the flower into leaflike structures. In each case, these mutations through Copyright Clearance Center, Inc.] CHAPTER 9 t Transcriptional Control of Gene Expression 355  Closed FIGURE 93 Overview of eukaryotic transcriptional control. Gene chromatin Inactive genes are assembled into regions of condensed chromatin “Off” that inhibit RNA polymerases and their associated general transcrip- tion factors from interacting with promoters. A pioneer transcription factor is able to bind to a specific regulatory sequence within the con- Repressors Pioneer densed chromatin and interact with chromatin-remodeling enzymes transcription and histone acetylases that decondense the chromatin, making it factors accessible to RNA polymerase II and the general transcription factors. Chromatin Additional activator proteins then bind to specific transcription- co-activators control elements in both promoter-proximal sites and distant enhanc- Ac Ac Ac Ac Ac ers, where they interact with one another and with the multisubunit Open chromatin Mediator complex to assemble RNA polymerase II (Pol II) and general transcription factors on promoters. Alternatively, repressor proteins bind to other transcription-control elements to inhibit transcrip- Me Me Me tion initiation by Pol II and interact with multiprotein co-repressor complexes to condense chromatin. During transcriptional activation, Repressors Activators Pol II initiates transcription, but pauses after transcribing fewer than 100 nucleotides due to the action of the elongation inhibitor NELF associated with DSIF. Activators promote the association of the Pol II-NELF-DSIF complex with elongation factor P-TEFb, which releases NELF and allows productive elongation through the gene. DSIF is the DRB sensitivity-inducing factor, NELF is the negative elongation factor, and P-TEFb is a protein kinase made up of CDK9 and cyclin T. Ac See S. Malik and R. G. Roeder, 2010, Nat. Rev. Genet. 11:761. Ac Me Me Me IIH Ac IID IIE IIH Ac IIB IIA IIF Me Pol II Mediator Activators, Activators another Ac Pol II Ac Pausing Gene P-TEFb “On” Scaffold Pol II Ac Scaffold Ac 7 IID NELF IID MeG Activators DSIF DSIF IIA IIA Me Me Nascent transcript Pol II 9.1 Control of Gene Expression in and how rapidly they are synthesized. When transcription of a gene is repressed, the corresponding mRNA and encoded Bacteria protein or proteins are synthesized at low rates. Conversely, Because the structure and function of a cell are determined when transcription of a gene is activated, both the mRNA by the proteins it contains, the control of gene expression and encoded protein or proteins are produced at much is a fundamental aspect of molecular cell biology. Most higher rates. commonly, the “decision” to transcribe the gene encoding In most bacteria and other single-celled organisms, gene a particular protein is the major mechanism for controlling expression is highly regulated in order to adjust the cell’s en- production of the encoded protein in a cell. By controlling zymatic machinery and structural components to changes in transcription, a cell can regulate which proteins it produces the nutritional and physical environment. Thus at any given 356 CHAPTER 9 t Transcriptional Control of Gene Expression time, a bacterial cell normally synthesizes only those proteins initiation (i.e., the number of times per minute that RNA that are required for its survival under the current condi- polymerases initiate transcription). The sequence shows the tions. Here we describe the basic features of transcriptional strand of DNA that has the same 5′→3′ orientation as the control in bacteria, using the lac operon and the glutamine transcribed RNA (i.e., the nontemplate strand). However, synthetase gene in E. coli and the xpt-pbuX operon in the σ70-RNA polymerase initially binds to double-stranded Bacillus subtilis as our primary examples. Many of the same DNA. After the polymerase transcribes a few tens of base features are involved in eukaryotic transcriptional control, pairs, σ70 is released. Thus σ70 acts as an initiation factor which will be the subject of the remainder of this chapter. that is required for transcription initiation, but not for RNA strand elongation once initiation has taken place. Transcription Initiation by Bacterial RNA Initiation of lac Operon Transcription Can Be Polymerase Requires Association with a Sigma Repressed or Activated Factor When E. coli is in an environment that lacks lactose, syn- In E. coli, about half the genes are clustered into operons, each thesis of lac mRNA is repressed so that cellular energy is of which encodes enzymes involved in a particular metabolic not wasted synthesizing enzymes the cell does not require. In pathway or proteins that interact to form one multisubunit an environment containing both lactose and glucose, E. coli protein complex. For instance, the trp operon discussed in cells preferentially metabolize glucose, the central molecule Chapter 5 encodes five polypeptides needed in the biosynthe- of carbohydrate metabolism. The cells metabolize lactose at sis of tryptophan (see Figure 5-13). Similarly, the lac operon a high rate only when lactose is present and glucose is largely encodes three proteins required for the metabolism of lactose, depleted from the medium. They achieve this metabolic a sugar present in milk. Because a bacterial operon is tran- adjustment by repressing transcription of the lac operon scribed from one start site into a single mRNA, all the genes until lactose is present and allowing synthesis of only low within an operon are coordinately regulated; that is, they are levels of lac mRNA until the cytosolic concentration of glu- all activated or repressed at the same time to the same extent. cose falls to low levels. Transcription of the lac operon under The transcription of operons, as well as that of isolated different conditions is controlled by lac repressor protein genes, is controlled by interplay between RNA polymerase and catabolite activator protein (CAP) (also called CRP, for and specific repressor and activator proteins. In order to ini- cAMP receptor protein), each of which binds to a specific tiate transcription, E. coli RNA polymerase must associate DNA sequence in the lac transcription-control region; these with one of a small number of σ (sigma) factors. The most two sequences are called the operator and the CAP site, common one in eubacterial cells is σ70. This σ-factor binds to respectively (Figure 9-4, top). both RNA polymerase and promoter DNA sequences, bring- For transcription of the lac operon to begin, the σ70 sub- ing the RNA polymerase enzyme to the promoter. It recog- unit of the RNA polymerase must bind to the lac promoter nizes and binds to both a six-base-pair sequence centered at the −35 and −10 promoter sequences. When no lactose at about 10 bp and a seven-base-pair sequence centered at is present, the lac repressor binds to the lac operator, which about 35 bp upstream from the +1 transcription start. Con- overlaps the transcription start site. Therefore, the lac re- sequently, the −10 sequence and the −35 sequence together pressor bound to the operator site blocks σ70 binding and constitute a promoter for E. coli RNA polymerase associ- hence transcription initiation by RNA polymerase (Figure ated with σ70 (see Figure 5-10b). Although the promoter 9-4a). When lactose is present, it binds to specific binding sequences contacted by σ70 are located at −35 and −10, sites in each subunit of the tetrameric lac repressor, causing E. coli RNA polymerase binds to the promoter-region DNA a conformational change in the protein that makes it dis- from roughly −50 to +20 through interactions with DNA sociate from the lac operator. As a result, the polymerase that do not depend on the sequence. The σ-factor also assists can bind to the promoter and initiate transcription of the the RNA polymerase in separating the DNA strands at the lac operon. However, when glucose is also present, the fre- transcription start site and in inserting the coding strand quency of transcription initiation is very low, resulting in into the active site of the polymerase so that transcription the synthesis of only low levels of lac mRNA and thus of starts at +1 (see Figure 5-11, step 2 ). The optimal σ70-RNA the proteins encoded by the lac operon (Figure 9-4b). The polymerase promoter sequence, determined as the “consensus frequency of transcription initiation is low because the −35 sequence” of multiple strong promoters, is and −10 sequences in the lac promoter differ from the ideal σ70-binding sequences shown previously. −35 region −10 region Once glucose is depleted from the medium and the intracel- ACA——15–17 bp——AA lular glucose concentration falls, E. coli cells respond by syn- thesizing cyclic AMP (cAMP). As the concentration of cAMP This consensus sequence shows the most commonly occur- increases, it binds to a site in each subunit of the dimeric CAP ring base at each of the positions in the −35 and −10 re- protein, causing a conformational change that allows the pro- gions. The size of the font indicates the importance of the tein to bind to the CAP site in the lac transcription-control base at that position, as determined by the influence of region. The bound CAP-cAMP complex interacts with the mutations of these bases on the frequency of transcription polymerase bound to the promoter, greatly increasing the 9.1 Control of Gene Expression in Bacteria 357  1 (transcription start site) frequency of transcription initiation. This activation leads to Promoter lacZ synthesis of high levels of lac mRNA and subsequently of the CAP site Operator enzymes encoded by the lac operon (Figure 9-4c). E. coli lac transcription-control regions In fact, the lac operon is more complex than depicted X70 in the simplified model in Figure 9-4a–c. The tetrameric lac Pol repressor actually binds to two DNA sequences simultane- CAP (a) ously, one at the primary operator (lacO1), which over- lac repressor laps the region of DNA bound by RNA polymerase at the  lactose  glucose lacZ promoter, and the other at one of two secondary operators (low cAMP) No mRNA transcription centered at +412 (lacO2), within the lacZ protein-coding lactose region, and −82 (lacO3) (Figure 9-4d). The lac repressor tetramer is a dimer of dimers. Each dimer binds to one op- (b) erator (Figure 9-4d). Simultaneous binding of the tetrameric  lactose X70 lacZ lac repressor to the primary lac operator and one of the two  glucose Pol (low cAMP) Low transcription secondary operators is possible because DNA is quite flex- ible, as we saw in the wrapping of DNA around the surface of a histone octamer in the nucleosomes of eukaryotes (see (c) cAMP Figure 8-24). The secondary operators function to increase X70 the local concentration of lac repressor in the micro-vicinity  lactose  glucose Pol lacZ of the primary operator where repressor binding blocks RNA High transcription (high cAMP) polymerase binding. Since the equilibrium of binding reac- tions depends on the concentrations of the binding partners, the resulting increased local concentration of lac repressor in (d) O3 O1 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 other- wise 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 Lac repressor repres of 70. Mutation of only O2 or only O3 reduces repression Promoter lacZ twofold, indicating that either one of these secondary opera- Promoter O3 O1 O2 O3 O1 O2 tors can provide most of the increase in repression. lacZ Although the promoters for different E. coli genes exhibit Lac repressor considerable homology, their exact sequences differ. The pro- moter sequence determines the intrinsic frequency at which FIGURE 94 Regulation of transcription from the lac operon of RNA polymerase–σ complexes initiate transcription of a gene E. coli. (Top) The transcription-control region, composed of roughly in the absence of a repressor or activator protein. Promoters a hundred base pairs, includes three protein-binding regions: the CAP site, which binds catabolite activator protein; the lac promoter, that support a high frequency of transcription initiation have which binds the σ70-RNA polymerase complex; and the lac opera- −10 and −35 sequences similar to the ideal promoter shown tor, which binds lac repressor. The lacZ gene encoding the enzyme previously and are called strong promoters. Those that support β-galactosidase, the first of the three genes in the operon, is shown to a low frequency of transcription initiation differ from this ideal the right. (a) In the absence of lactose, very little lac mRNA is produced sequence and are called weak promoters. The lac operon, for because the lac repressor binds to the operator, inhibiting transcription instance, has a weak promoter whose sequence differs from the initiation by σ70-RNA polymerase. (b) In the presence of glucose and consensus strong promoter at several positions. Its low intrin- lactose, lac repressor binds lactose and dissociates from the operator, sic frequency of initiation is further reduced by the lac repres- allowing σ70-RNA polymerase to initiate transcription at a low rate. sor and substantially increased by the cAMP-CAP complex. (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 Small Molecules Regulate Expression of Many complex, which binds to the CAP site, where it interacts with RNA poly- Bacterial Genes via DNA-Binding Repressors and merase to increase the rate of transcription initiation. (d) The tetrameric Activators lac repressor binds to the primary lac operator (O1) and one of two sec- ondary operators (O2 or O3) simultaneously. The two structures are in Transcription of most E. coli genes is regulated by processes equilibrium. See B. Muller-Hill, 1998, Curr. Opin. Microbiol. 1:145. [Part (d) similar to those described for the lac operon, although the de- data from M. Lewis et al., 1996, Science 271:1247-1254, PDB IDs 1lbh and tailed interactions differ at each promoter. The general mech- 1lbg; and R. Daber et al., 2007, J. Mol. Biol. 370:609-619, PDB ID 2pe5.] anism involves a specific repressor that binds to the operator 358 CHAPTER 9 t Transcriptional Control of Gene Expression region of a gene or operon, thereby blocking transcription alternative sigma factors that recognize different consensus initiation. A small-molecule ligand binds to the repressor promoter sequences than σ70 does (Table 9-1). These alter- controlling its DNA-binding activity, and consequently the native σ-factors are required for the transcription of sets of frequency of transcription initiation and therefore the rate of genes with related functions, such as those involved in the synthesis of the mRNA and encoded proteins as appropriate response to heat shock or nutrient deprivation, motility, for the needs of the cell. As for the lac operon, many eubac- or sporulation in gram-positive eubacteria. In E. coli, there terial transcription-control regions contain one or more sec- are 6 alternative σ-factors in addition to the major “house- ondary operators that contribute to the level of repression. keeping” σ-factor, σ70. The genome of the gram-positive, Specific activator proteins, such as CAP in the lac op- sporulating bacterium Streptomyces coelicolor encodes 63 eron, also control transcription of a subset of bacterial genes σ-factors, the current record, based on sequence analysis of that have binding sites for the activator. Like CAP, other hundreds of eubacterial genomes. Most are structurally and activators bind to DNA together with RNA polymerase, functionally related to σ70. Transcription initiation by RNA stimulating transcription from a specific promoter. The polymerases containing σ70-like factors is regulated by re- DNA-binding activity of an activator can be modulated in pressors and activators that bind to DNA near the region response to cellular needs by the binding of specific small- where the polymerase binds. But one class, represented in molecule ligands (e.g., cAMP) or by post-translational E. coli by σ54, is unrelated to σ70 and functions differently. modifications, such as phosphorylation, that alter the con- formation of the activator. Transcription by σ54-RNA Polymerase Is Controlled by Activators That Bind Far from the Transcription Initiation from Some Promoters Promoter Requires Alternative Sigma Factors The sequence of σ54 is distinctly different from that of all the 70 Most E. coli promoters interact with σ -RNA polymerase, σ70-like factors. Transcription of genes by RNA polymer- the major initiating form of the bacterial enzyme. The ases containing σ54 is regulated solely by activators whose transcription of certain groups of genes, however, is initi- binding sites in DNA, referred to as enhancers, are generally ated by E. coli RNA polymerases containing one of several located 80–160 bp upstream from the transcription start site. TABLE 91 Sigma Factors of E. coli Promoter Consensus Sigma Factor Promoters Recognized −35 Region −10 Region σ70 (σD) Housekeeping genes, most genes in TTGACA TATAAT exponentially replicating cells σS (σ38) Stationary-phase genes and general TTGACA TATAAT stress response σ32 (σH) Induced by unfolded proteins in the TCTCNCCCTTGAA CCCCATNTA cytoplasm; genes encoding chaperones that refold unfolded proteins and protease systems leading to the degradation of unfolded proteins in the cytoplasm σE (σ24) Activated by unfolded proteins in the GAACTT TCTGA periplasmic space and cell membrane; genes encoding proteins that restore integrity to the cellular envelope σF (σ28) Genes involved in flagellum assembly CTAAA CCGATAT FecI (σ ) 18 Genes required for iron uptake TTGGAAA GTAATG −24 Region −12 Region σ 54 N (σ ) Genes for nitrogen metabolism and other CTGGNA TTGCA functions Data from T. M. Gruber and C. A. Gross, 2003, Annu. Rev. Microbiol. 57:441, and B. K. Cho et al., 2014, BMC Biol. 12:4. 9.1 Control of Gene Expression in Bacteria 359 Even when enhancers are moved more than a kilobase away later in this chapter, this activation mechanism resembles from a start site, σ54-activators can activate transcription. the predominant mechanism of transcriptional activation The best-characterized σ54-activator—the NtrC protein in eukaryotes. (nitrogen regulatory protein C)—stimulates transcription NtrC has ATPase activity, and ATP hydrolysis is re- of the glnA gene. The glnA gene encodes the enzyme glu- quired for activation of bound σ 54-RNA polymerase by tamine synthetase, which synthesizes the amino acid glu- phosphorylated NtrC. Mutants with an NtrC that is defec- tamine, the central molecule of nitrogen metabolism, from tive in ATP hydrolysis are invariably defective in stimulating glutamic acid and ammonia. The σ 54-RNA polymerase the σ54-RNA polymerase to melt the DNA strands at the binds to the glnA promoter but does not melt the DNA transcription start site. It is postulated that ATP hydrolysis strands and initiate transcription until it is activated by supplies the energy required for melting the DNA strands. In NtrC, a dimeric protein. NtrC, in turn, is regulated by a contrast, the σ70-polymerase does not require ATP hydroly- protein kinase called NtrB. In response to low levels of sis to separate the strands at a start site. glutamine, NtrB phosphorylates dimeric NtrC, which then binds to an enhancer upstream of the glnA promoter. Many Bacterial Responses Are Controlled by Enhancer-bound phosphorylated NtrC then stimulates the σ54-polymerase bound at the promoter to separate the Two-Component Regulatory Systems DNA strands and initiate transcription. As we have just seen, control of the E. coli glnA gene depends Electron microscopy studies have shown that phosphor- on two proteins, NtrC and NtrB. Such two-component regu- ylated NtrC bound at enhancers and σ54-polymerase bound latory systems control many responses of bacteria to changes at the promoter interact directly, forming a loop in the in their environment. At high concentrations of glutamine, DNA between the binding sites (Figure 9-5). As discussed glutamine binds to a sensor domain of NtrB, causing a NtrC dimers - (a) Pair of phosphorylated NtrC dimers - P P P P Enhancer glnA (–140 and promoter –108) (b) P P P P NtrC dimers - EXPERIMENTAL FIGURE 95 DNA looping permits interaction (b) Drawing (left) and electron micrograph (right) of the same fragment of bound NtrC and σ54-RNA polymerase. (a) Drawing (left) and preparation, showing NtrC dimers and σ54-RNA polymerase bound to electron micrograph (right) of DNA restriction fragment with phosphor- each other, with the intervening DNA forming a loop between them. ylated NtrC dimers bound to the enhancer region near one end and See W. Su et al., 1990, Proc. Natl. Acad. Sci. USA 87:5504. [Micrographs σ54-RNA polymerase bound to the glnA promoter near the other end. courtesy Harrison Echols and Carol Gross.] 360 CHAPTER 9 t Transcriptional Control of Gene Expression conformational change in the protein that inhibits its his- In each of these regulatory systems, one protein, called a his- tidine kinase activity (Figure 9-6a). At the same time, the tidine kinase sensor, contains a latent histidine kinase trans- regulatory domain of NtrC blocks its DNA-binding domain mitter domain that is regulated in response to environmental from binding the glnA enhancers. At low concentrations of changes detected by a sensor domain. When activated, the glutamine, glutamine dissociates from the sensor domain in transmitter domain transfers the γ-phosphate of ATP to a the NtrB protein, leading to activation of a histidine kinase histidine residue in the transmitter domain. The second pro- transmitter domain in NtrB that transfers the γ-phosphate tein, called a response regulator, contains a receiver domain of ATP to a histidine residue (H) in the transmitter do- homologous to the region of NtrC containing the aspartic main. This phosphohistidine then transfers the phosphate acid residue that is phosphorylated by activated NtrB. The to an aspartic acid residue (D) in the NtrC protein. This response regulator contains a second functional domain that causes a conformational change in NtrC that unmasks the is regulated by phosphorylation of the receiver domain. In NtrC DNA-binding domain so that it can bind to the glnA many cases, this domain of the response regulator is a enhancers. sequence-specific DNA-binding domain that binds to re- Many other bacterial responses are regulated by two lated DNA sequences and functions either as a repressor, proteins with homology to NtrB and NtrC (Figure 9-6b). 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 (a) Two-component system regulating response to low Gln controlling the direction in which the bacterium swims in NtrB NtrC response to a concentration gradient of nutrients. Although Regulatory Sensor domain domain all transmitter domains are homologous (as are receiver do- High [Gln] Gln D mains), the transmitter domain of a specific sensor protein H will phosphorylate only the receiver domains of specific re- sponse regulators, allowing specific responses to different en- His kinase DNA-binding transmitter domain vironmental changes. Similar two-component histidyl-aspartyl domain phospho-relay regulatory systems are also found in plants. Low [Gln] Sensor His kinase DNA-binding domain transmitter domain domain H D Expression of Many Bacterial Operons Is P P Controlled by Regulation of Transcriptional ATP ADP glnA enhancer Elongation In addition to regulation of transcription initiation by activa- tors and repressors, expression of many bacterial operons is (b) General two-component signaling system controlled by regulation of transcriptional elongation in the Sensor Receiver promoter-proximal region. This mechanism of control was domain domain first discovered in studies of trp operon transcription in E. coli Histidine D kinase H Response (see Figure 5-13). Transcription of the trp operon is repressed sensor regulator by the trp repressor when the concentration of tryptophan in His kinase Effector the cytoplasm is high. But the low level of transcription initia- domain domain Stimulus tion that still occurs is further controlled by a process called Sensor His kinase attenuation when the concentration of charged tRNATrp is suf- domain domain ficient to support a high rate of protein synthesis. The first H D 140 nt of the trp operon does not encode proteins required for P ATP P Effector tryptophan biosynthesis, but rather consists of a short peptide domain “leader sequence,” as diagrammed in Figure 9-7a. Region 1 ADP of this leader sequence contains two successive Trp codons. Region 3 can base-pair with either region 2 or region 4. A Response ribosome follows closely behind the RNA polymerase, initiat- FIGURE 96 Two-component regulatory systems. (a) At low ing translation of the leader peptide shortly after the 5′ end of cytoplasmic concentrations of glutamine, glutamine dissociates from the trp leader sequence emerges from the RNA polymerase. NtrB, resulting in a conformational change that activates a protein When the concentration of tRNATrp is sufficient to support a kinase transmitter domain that transfers an ATP γ-phosphate to a high rate of protein synthesis, the ribosome translates quickly conserved histidine (H) in the transmitter domain. This phosphate is then transferred to an aspartic acid (D) in the regulatory domain of through region 1 into region 2, blocking the ability of region the response regulator NtrC. This converts NtrC into its activated form, 2 to base-pair with region 3 as it emerges from the surface which binds the enhancer sites upstream of the glnA promoter (see of the transcribing RNA polymerase (Figure 9-7b, left). In- Figure 9-5). (b) General organization of two-component histidyl-aspartyl stead, region 3 base-pairs with region 4 as soon as it emerges phospho-relay regulatory systems in bacteria and plants. See A. H. West from the surface of the polymerase, forming a stem-loop (see and A. M. Stock, 2001, Trends Biochem. Sci. 26:369. Figure 5-9a) followed by several uracils, which is a signal for 9.1 Control of Gene Expression in Bacteria 361  (a) trp leader RNA Translation start codon 1 50 100 140 | | || | 5’| 1 2 3 4 UUUUU| 3’ (b) Translation of trp leader High tryptophan Low tryptophan Ribosome covers region 2 Ribosome is stalled at trp codons in region 1 Leader peptide Leader peptide 2-3 stem-loop 2 3 forms 2 1 UUUUU 3’ 5’ RNA polymerase 3-4 stem-loop 4 1 continues 3 4 RNA polymerase forms transcription terminates transcription 5’ FIGURE 97 Transcriptional control by regulation of RNA At high concentrations of charged tRNATrp, formation of the 3–4 stem- polymerase elongation and termination in the E. coli trp operon. loop followed by a series of uracils causes termination of transcription. (a) Diagram of the 140-nucleotide trp leader RNA. The numbered At low concentrations of charged tRNATrp, region 3 is sequestered in regions are critical to attenuation. (b) Translation of the trp leader the 2–3 stem-loop and cannot base-pair with region 4. In the absence sequence begins near the 5′ end soon after it is transcribed, while tran- of the stem-loop structure required for termination, transcription of the scription of the rest of the polycistronic trp mRNA molecule continues. trp operon continues. See C. Yanofsky, 1981, Nature 289:751. bacterial RNA polymerase to pause transcription and termi- not bound by the aptamers, and alternative RNA structures nate. As a consequence, the remainder of the long trp operon is form that do not induce transcription termination, allowing not transcribed, and the cell does not waste the energy required transcription of genes encoding enzymes involved in the syn- for tryptophan synthesis, or for the translation of the encoded thesis of the metabolites. As we will see below, although the proteins, when the concentration of tryptophan is high. mechanism in eukaryotes is different, regulation of promoter- However, when the concentration of tRNA Trp is not proximal transcriptional pausing and termination has recently sufficient to support a high rate of protein synthesis, the been discovered to occur frequently in the regulation of gene ribosome stalls at the two successive Trp codons in region 1 expression in multicellular organisms as well. (Figure 9-7b, 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 KEY CONCEPTS OF SECTION 9.1 with region 4, so the 3–4 hairpin does not form and does not cause RNA polymerase pausing or transcription termi- Control of Gene Expression in Bacteria nation. As a result, the proteins required for tryptophan syn- thesis are translated by ribosomes that initiate translation r Gene expression in both prokaryotes and eukaryotes is regu- at the start codons for each of these proteins in the long lated primarily by mechanisms that control gene transcription. polycistronic trp mRNA. r The first step in the initiation of transcription in E. coli Attenuation of transcription elongation also occurs at some is the binding of a σ-factor complexed with an RNA poly- operons and single genes encoding enzymes involved in the merase to a promoter. biosynthesis of other amino acids and metabolites through the r The nucleotide sequence of a promoter determines its function of riboswitches. Riboswitches are sequences of RNA strength, that is, how frequently different RNA polymerase most commonly found in the 5′ untranslated region of bacte- molecules can bind and initiate transcription per minute. rial mRNAs. They fold into complex tertiary structures called aptamers that bind small-molecule metabolites when those r Repressors are proteins that bind to operator sequences that metabolites are present at sufficiently high concentrations. In overlap or lie adjacent to promoters. Binding of a repressor to some cases, this binding results in the formation of stem-loop an operator inhibits transcription initiation or elongation. structures that lead to early termination of transcription, as in r The DNA-binding activity of most bacterial repressors is the Bacillus subtilis xpt-pbuX operon, which encodes enzymes modulated by small-molecule ligands. This allows bacterial involved in purine synthesis (Figure 9-8). When the concentra- cells to regulate transcription of specific genes in response tion of small-molecule metabolites is lower, the metabolites are 362 CHAPTER 9 t Transcriptional Control of Gene Expression (a) (b) Folding of aptamer Gene “On” Transcription continues Low purine concentration 5’ Pol High purine concentration Transcription termination UUUUU 3’ Purine 5’ Gene “Off” 5’ FIGURE 98 Riboswitch control of transcription termination in tryptophan concentrations (see Figure 9-7), i.e., a stem loop followed by B. subtilis. (a) During transcription of the Bacillus subtilis xpt-pbuX a run of Us. At low purine concentrations, an alternative RNA structure operon, which encodes enzymes involved in purine synthesis, the 5′ forms that prevents formation of the transcription termination signal, untranslated region of the mRNA can fold into alternative structures permitting transcription of the operon. Note the alternative base pair- depending on the concentration of purines in the cytoplasm, forming ing of the red and blue regions of the RNA. (b) Structure of the purine the “purine riboswitch.” At high concentrations of purines, the riboswitch riboswitch bound to a purine (cyan) as determined by X-ray crystallog- folds into an aptamer that binds a purine ligand (cyan circle), allowing raphy. See A. D. Garst, A. L. Edwards, and R. T. Batey, 2011, Cold Spring formation of a stem-loop transcription termination signal similar to the Harb. Perspect. Biol. 3:a003533. [Part (b) data from R. T. Batey, S. D. Gilbert, and termination signal that forms in the E. coli trp operon mRNA at high R. K. Montagne, 2004, Nature 432:411, PDB ID 4fe5.] to changes in the concentration of various nutrients in the γ-phosphate of an ATP is transferred first to a histidine in the environment and metabolites in the cytoplasm. sensor protein and then to an aspartic acid in a second protein, r The lac operon and some other bacterial genes are also the response regulator. The phosphorylated response regulator regulated by activator proteins that bind next to a promoter then performs a specific function in response to the stimulus, and increase the frequency of transcription initiation by such as binding to DNA regulatory sequences, thereby stimulat- interacting directly with RNA polymerase bound to that ing or repressing transcription of specific genes (see Figure 9-6). promoter. r Transcription in bacteria can also be regulated by control 70 r The major sigma factor in E. coli is σ , but several other, of transcriptional elongation in the promoter-proximal re- less abundant sigma factors are also found, each recogniz- gion. This control can be exerted by ribosome binding to the ing different consensus promoter sequences or interacting nascent mRNA, as in the case of the E. coli trp operon (see with different activators. Figure 9-7), or by riboswitches, RNA sequences that bind small molecules, as for the B. subtilis xpt-pbuX operon (see r Transcription initiation by all E. coli RNA polymerases, Figure 9-8), to determine whether a stem-loop followed by except those containing σ54, can be regulated by repressors a string of uracils forms, causing the bacterial RNA poly- and activators that bind near the transcription start site (see merase to pause and terminate transcription. Figure 9-4). r Genes transcribed by σ54-RNA polymerase are regulated by activators that bind to enhancers located about 100 base pairs upstream from the start site. When the activator and σ54-RNA polymerase interact, the DNA between their 9.2 Overview of Eukaryotic Gene Control binding sites forms a loop (see Figure 9-5). In bacteria, gene control serves mainly to allow a single cell r In two-component regulatory systems, one protein acts as to adjust to changes in its environment so that its growth and a sensor, monitoring the level of nutrients or other compo- division can be optimized. In multicellular organisms, envi- nents in the environment. Under appropriate conditions, the ronmental changes also induce changes in gene expression. An example is the response to low oxygen concentrations 9.2 Overview of Eukaryotic Gene Control 363 (hypoxia), in which a specific set of genes is rapidly induced Regulatory Elements in Eukaryotic DNA Are that helps the cell survive under the hypoxic conditions. Found Both Close to and Many Kilobases Away These genes include those encoding secreted angiogenic proteins that stimulate the growth and penetration of new from Transcription Start Sites capillaries into the surrounding tissue. However, the most Direct measurements of the transcription rates of multiple characteristic and biologically far-reaching purpose of gene genes in different cell types have shown that regulation control in multicellular organisms is execution of the genetic of transcription, either at the initiation step or during program that underlies embryological development. Genera- elongation in the promoter-proximal region, is the most tion of the many different cell types that collectively form widespread form of gene control in eukaryotes, as it is in a multicellular organism depends on the right genes being bacteria. In eukaryotes, as in bacteria, a DNA sequence activated in the right cells at the right time during the devel- that specifies where RNA polymerase binds and initiates opmental period. transcription of a gene is called a promoter. Transcription In most cases, once a developmental step has been from a particular promoter is controlled by DNA-binding taken by a cell, it is not reversed. Thus these decisions proteins that are functionally equivalent to bacterial re- are fundamentally different from the reversible activation pressors and activators. However, eukaryotic transcrip- and repression of bacterial genes in response to environ- tional regulatory proteins can often function either to mental conditions. In executing their genetic programs, activate or to repress transcription, depending on their many differentiated cells (e.g., skin cells, red blood cells, associations with other proteins. Consequently, they are and antibody-producing cells) march down a pathway to more generally called transcription factors. final cell death, leaving no progeny behind. The fixed pat- The DNA control elements in eukaryotic genomes to terns of gene control leading to differentiation serve the which transcription factors bind are often located much needs of the whole organism and not the survival of an farther from the promoter they regulate than is the case individual cell. in bacterial genomes. In some cases, transcription factors Despite the differences in the purposes of gene con- bind at regulatory sites tens of thousands of base pairs ei- trol in bacteria and eukaryotes, two key features of ther upstream (opposite to the direction of transcription) or transcriptional control first discovered in bacteria and downstream (in the same direction as transcription) from the described in the previous section also apply to eukary- promoter. As a result of this arrangement, transcription of a otic cells. First, protein-binding regulatory DNA se- single gene may be regulated by the binding of multiple dif- quences, or transcription-control regions, are associated ferent transcription factors to alternative control elements, with genes. Second, specific proteins that bind to a gene’s which direct expression of the same gene in different types of transcription-control regions determine where transcrip- cells and at different times during development. tion will start and either activate or repress transcription. For example, several separate transcription-control re- One fundamental difference between transcriptional con- gions regulate expression of the mammalian gene encoding trol in bacteria and in eukaryotes is a consequence of the the transcription factor Pax6. As mentioned in Chapter 1, association of eukaryotic chromosomal DNA with histone Pax6 protein is required for development of the eye. Pax6 octamers, forming nucleosomes that associate into chro- is also required for the development of certain regions of matin fibers that further associate into chromatin of vary- the brain and spinal cord, and the cells in the pancreas that ing degrees of condensation (see Figures 8-24, 8-25, 8-27, secrete hormones such as insulin. As also mentioned in and 8-28). Eukaryotic cells exploit chromatin structure Chapter 1, heterozygous humans with only one functional to regulate transcription, a mechanism of transcriptional Pax6 gene are born with aniridia, a lack of irises in the eyes control that is not available to bacteria. In multicellular (see Figure 1-30d). In mammals, the Pax6 gene is expressed eukaryotes, many inactive genes are assembled into con- from at least three alternative promoters that function in dif- densed chromatin, which inhibits binding of the RNA ferent cell types and at different times during embryogenesis polymerases and general transcription factors required (Figure 9-9a). for transcription initiation (see Figure 9-3). Activator pro- Researchers often analyze transcription-control regions by teins, which bind to transcription-control regions near the preparing recombinant DNA molecules that combine a frag- transcription start site of a gene as well as kilobases away, ment of DNA to be tested with the coding region for a reporter promote chromatin decondensation, binding of RNA gene whose expression is easily assayed. Typical reporter genes polymerase to the promoter, and transcriptional elonga- include the gene that encodes luciferase, an enzyme that gener- tion. Repressor proteins, which bind to alternative control ates light that can be assayed with great sensitivity and over elements, cause condensation of chromatin and inhibition many orders of magnitude of intensity using a luminometer. of polymerase binding or elongation. In this section, we Other frequently used reporter genes encode green fluorescent discuss the general principles of eukaryotic gene control protein (GFP), which can be visualized by fluorescence micros- and point out some similarities and differences between copy (see Figures 4-9d and 4-16), and E. coli β-galactosidase, bacterial and eukaryotic systems. Subsequent sections of which generates an intensely blue insoluble precipitate when in- this chapter will address specific aspects of eukaryotic cubated with the colorless soluble lactose analog X-gal. When transcription in greater detail. transgenic mice (see Figure 6-40) containing a β-galactosidase 364 CHAPTER 9 t Transcriptional Control of Gene Expression  (a) AAA 0 12 3 4 α 5 6 7 8 9 10 11 12 13 Pancreas Lens and Telencephalon Retina Retina Di- and rhombo- cornea encephalon Transcript a 0 2 3 4 5 6 7 8 9 10 11 12 13 AAA Transcript b 1 2 3 4 5 6 7 8 9 10 11 12 13 AAA Transcript c α 5 6 7 8 9 10 11 12 13 5 10 15 20 25 30 kb (b) (c) LP P (d) PAX6 0 100 200 300 500 kb RCN1 ELP4 FIGURE 99 Transcription-control regions of the mouse Pax6 below the line representing this region of human DNA. PAX6 exons are gene and the orthologous human PAX6 gene. (a) Three alternative diagrammed as red rectangles above the line. The three PAX6 promoters Pax6 promoters are used at distinct times during embryogenesis in first characterized in the mouse are shown by rightward arrowheads, different tissues of the developing mouse embryo. Transcription-control and the control regions shown in (a) are represented by gray rectangles. regions regulating expression of Pax6 in different tissues are indicated Regions flanking the gene where the sequence is partially conserved by colored rectangles. These control regions are some 200–500 bp in in most vertebrates (as in Figure 9-10a) are shown as ovals. Colored length. (b) Expression of a β-galactosidase reporter transgene fused ovals represent sequences that cause expression of the transgene in to the 8 kb of mouse DNA upstream from exon 0. A transgenic mouse specific neuroanatomical locations in the zebrafish central nervous embryo 10.5 days after fertilization was stained with X-gal to reveal system. Ovals with the same color stimulated expression in the same β-galactosidase. Lens pit (LP) is the tissue that will develop into the lens region. Gray ovals represent conserved sequences that did not stimulate of the eye. Expression was also observed in tissue that will develop into reporter-gene expression in the developing zebrafish embryo, or were the pancreas (P). (c) Expression in a mouse embryo at 13.5 days after not tested. Such conserved regions may function only in combination, fertilization of a β-galactosidase reporter gene linked to the sequence or they may have been conserved for some reason other than regula- in part (a) between exons 4 and 5 marked Retina. Arrow points to tion of transcription, such as proper folding of the chromosome into nasal and temporal regions of the developing retina. (d) Human PAX6 topological domains (see Figure 8-34). [Part (a) data from B. Kammendal control regions identified in the 600-kb region of human DNA between et al., 1999, Devel. Biol. 205:79. Part (b) republished with permission of Elsevier, the upstream gene RCN1 and the promoter of the downstream ELP4 B. Kammendal et al., “Distinct cis-essential modules direct the time-space pattern gene. RCN1 and ELP4 are transcribed in the opposite direction from of the Pax6 gene activity,” Developmental Biology, 1999, 205(1): 79–97; permission PAX6, as represented by the leftward-pointing arrows associated with conveyed through Copyright Clearance Center, Inc. Part (c) courtesy of Peter Gruss their first exons. RCN1 and ELP1 exons are shown as black rectangles and Birgitta Kammandel. Part (d) data from S. Batia et al., 2014, Devel. Biol. 387:214.] 9.2 Overview of Eukaryotic Gene Control 365  reporter gene fused to 8 kb of DNA upstream from Pax6 exon 0 For example, there is a human DNA sequence, which were produced, β-galactosidase was observed in the developing is highly conserved between humans, mice, chickens, lens, cornea, and pancreas of the embryo halfway through ges- frog, and fish, about 500 kb downstream of the SALL1 tation (Figure 9-9b). Analysis of transgenic mice with smaller gene (Figure 9-10a). SALL1 encodes a transcription fac- fragments of DNA from this region allowed the mapping of the tor required for normal development of the limbs. When separate transcription-control regions regulating transcription transgenic mice were produced containing this conserved in the pancreas, and in both the lens and cornea. Transgenic DNA sequence linked to a β-galactosidase reporter gene mice with other reporter gene constructs revealed additional (Figure 9-10b), the transgenic embryos expressed a very transcription-control regions (see Figure 9-9a). These regions high level of β-galactosidase in the developing limb buds control transcription in the developing retina and in different (Figure 9-10c). Human patients with deletions in this regions of the developing brain (encephalon). Some of these region of the genome develop with limb abnormalities. transcription-control regions are in introns between exons 4 These results indicate that this conserved region directs and 5 and between exons 7 and 8. For example, a reporter transcription of the SALL1 gene in the developing limb. gene under control of the region labeled Retina in Figure 9-9a Presumably, other transcription-control regions control between exons 4 and 5 led to reporter-gene expression specifi- expression of this gene in other types of cells, where it cally in the retina (Figure 9-9c). functions in the normal development of the ears, the lower Control regions for many genes are found hundreds of intestine, and kidneys. kilobases away from the coding exons of the gene. One Because the sequences and functions of transcription- method for identifying such distant control regions is to control regions are often conserved through evolution, the compare the sequences of distantly related organisms. transcription factors that bind to these transcription-control Transcription-control regions for a conserved gene are also regions to regulate gene expression in specific cell types are often conserved and can be recognized in the background of presumably conserved during evolution as well. This has nonfunctional sequences that diverge during evolution. made it possible to assay control regions in human DNA by (a) Comparative analysis Sequence similarity to human Mouse FIGURE 910 The human SALL1 enhancer activates expression Chicken of a reporter gene in limb buds of the developing mouse embryo. (a) Graphic representation of the conservation of DNA sequence in a region of the human genome (in the interval of chromosome 16 from Frog 50214 kb to 50220.5 kb) about 500 kb downstream from the SALL1 gene, which encodes a zinc-finger transcription repressor. A region of roughly 500 bp of nonprotein-coding sequence is conserved from Fish zebrafish to human. Nine hundred base pairs of human DNA including 50215 50217 50219 Chromosome 16 (kb) this conserved region were inserted into a plasmid next to the coding region for E. coli β-galactosidase. (b) The plasmid was microinjected into a pronucleus of a fertilized mouse egg and implanted in the (b) Mouse egg microinjection (c) E11.5 reporter staining uterus of a pseudopregnant mouse to generate a transgenic mouse embryo with the reporter-gene-containing plasmid incorporated into its genome (see Figure 5-43). (c) After 11.5 days of development, at the time when limb buds develop, the fixed and permeabilized embryo was incubated in X-gal, which is converted by β-galactosidase into an insoluble, intensely blue compound. The results showed that Forelimb the conserved region contains an enhancer that stimulates strong bud transcription of the β-galactosidase reporter gene specifically in limb Hindlimb buds. [Part (a) data from A. Visel et al., 2007. VISTA Enhancer Browser—a bud database of tissue-specific human enhancers. Nucleic Acids Res. 35:D88–92. Part (b) ©Deco/Alamy. Part (c) republished with permission of Nature, from Pennacchio, L.A., et al., “In vivo enhancer analysis of human conserved non- coding sequences”, Nature, 444, 499–506, 2006; permission conveyed through Copyright Clearance Center, Inc.] 366 CHAPTER 9 t Transcriptional Control of Gene Expression reporter-gene expression in transgenic zebrafish, a proce- [NaCl] dure that is far simpler, faster, and less expensive than pre- paring transgenic mice (Figure 9-9d). After discussing the Pol I proteins that function with RNA polymerase to carry out transcription in eukaryotic cells and eukaryotic promoters, we Total will return to a discussion of how such distant transcription- RNA synthesis in presence protein control regions, called enhancers, are thought to function. of 1 μg/ml α-amanitin Pol II Three Eukaryotic RNA Polymerases Catalyze RNA synthesis Formation of Different RNAs The nuclei of all eukaryotic cells examined so far (e.g., verte- Protein brate, Drosophila, yeast, and plant cells) contain three differ- Pol III ent RNA polymerases, designated I, II, and III. These enzymes are eluted at different salt concentrations during ion-exchange 10 20 30 40 50 chromatography, reflecting the differences in their net charges. Fraction number The three nuclear RNA polymerases also differ in their sensi- tivity to α-amanitin, a poisonous cyclic octapeptide produced EXPERIMENTAL FIGURE 911 Liquid chromatography sepa- by some mushrooms (Figure 9-11). RNA polymerase I is insen- rates and identifies the three eukaryotic RNA polymerases, each sitive to α-amanitin, but RNA polymerase II is very sensitive— with its own sensitivity to -amanitin. A protein extract from the nu- clei of cultured eukaryotic cells was passed through a DEAE Sephadex the drug binds near the active site of the enzyme and inhibits column and adsorbed protein eluted (black curve) with a solution of translocation of the enzyme along the DNA template. RNA constantly increasing NaCl concentration. An aliquot of each fraction of polymerase III has intermediate sensitivity. eluate collected from the column was assayed for RNA polymerase ac- Each eukaryotic RNA polymerase catalyzes transcription tivity without (red curve) and with (green shading) 1 μg/ml α-amanitin. of genes encoding different classes of RNA (Table 9-2). RNA This concentration of α-amanitin inhibits polymerase II activity but polymerase I (Pol I), located in the nucleolus, transcribes has no effect on polymerases I and III. Polymerase III is inhibited by genes encoding precursor rRNA (pre-rRNA), which is pro- 10 μg/ml of α-amanitin, whereas polymerase I is unaffected even at cessed into 28S, 5.8S, and 18S rRNAs. RNA polymerase III this higher concentration. See R. G. Roeder, 1974, J. Biol. Chem. 249:241. (Pol III) transcribes genes encoding tRNAs, 5S rRNA, and an array of small stable RNAs, including one involved in RNA splicing (U6) and the RNA component of the signal recog- also produces four of the five small nuclear RNAs (snRNAs) nition particle (SRP) involved in directing nascent proteins that take part in RNA splicing and micro-RNAs (miRNAs) to the endoplasmic reticulum (see Chapter 13). RNA poly- involved in translation control, as well as the closely re- merase II (Pol II) transcribes all protein-coding genes: that lated endogenous small interfering RNAs (siRNAs) (see is, it functions in production of mRNAs. RNA polymerase II Chapter 10). TABLE 92 Classes of RNA Transcribed by the Three Eukaryotic Nuclear RNA Polymerases and Their Functions Polymerase RNA Transcribed RNA Function RNA polymerase I Pre-rRNA (28S, 18S, 5.8S rRNAs) Ribosome components, protein synthesis RNA polymerase II mRNA Encodes protein snRNAs RNA splicing siRNAs Chromatin-mediated repression, translation control miRNAs Translation control RNA polymerase III tRNAs Protein synthesis 5S rRNA Ribosome component, protein synthesis snRNA U6 RNA splicing 7S RNA