Mechanisms of Transcription
Page 689 Figure 16.10 Structures of theophylline and caffeine. Although these compounds differ only by a single methyl group, a specific synthetic RNA can bind to theophylline 10,000fold more tightly than to caffeine. Figure 16.10). Theophylline is used in the treatment of chronic asthma but the level must be carefully controlled to avoid side effects. The monitoring of theophylline by conventional antibodybased clinical chemistry is difficult because caffeine and theophylline differ only by a single methyl group. Therefore antitheophylline antibodies show considerable crossreaction with caffeine. RNA molecules have been found that bind theophylline 10,000fold more tightly than caffeine. Other extensions of the technology have used selection procedures to identify new, synthetic ribozymes and potential therapeutic RNAs. 16.4— Mechanisms of Transcription The Initial Process of RNA Synthesis Is Transcription The process by which RNA chains are made from DNA templates is called transcription. All known transcription reactions take the following form: Enzymes that catalyze this reaction are designated RNA polymerases; it is important to recognize that they are absolutely template dependent. In contrast to DNA polymerases, however, RNA polymerases do not require a primer molecule. The energetics favoring the RNA polymerase reaction are twofold: first, the 5 a nucleotide phosphate of the ribonucleoside triphosphate is converted from a phosphate anhydride to a phosphodiester bond with a change in free energy ( G ) of approximately 3 kcal (12.5 kJ) mol–1 under standard conditions; second, the released pyrophosphate, PPi, can be cleaved into two phosphates by pyrophosphatase so that its concentration is low and phosphodiester bond formation is more favored relative to standard conditions (see Chapter 6 for a fuller discussion of metabolic coupling). Since a DNA template is required for RNA synthesis, eukaryotic transcription takes place in the cell nucleus or mitochondrial matrix. Within the nucleus, the nucleolus is the site of rRNA synthesis, whereas mRNA and tRNA are synthesized in the nucleoplasm. Prokaryotic transcription is accomplished on the cell's DNA, which is located in a relatively small region of the cell. In the case of prokaryotic plasmids, the DNA template need not be associated with the chromosome. Structural changes occur in DNA during its transcription. In the polytene chromosomes of Drosophila, transcriptionally active genes are visualized in the light microscope as puffs distinct from the condensed, inactive chromatin. Furthermore, the nucleosome patterns of active genes are disrupted so that active chromatin is more accessible to, for example, DNase attack. In prokaryotes and eukaryotes, the DNA double helix is transiently opened (unwound) as the transcription complex proceeds down the DNA. These openings and unwindings are a manifestation of a topological necessity. If the RNA chain were copied off DNA without this unwinding, the transcription complex and growing end of the RNA chain would have to wind around the double helix once every 10 base pairs as they travel from the beginning of the gene to its end. Such a process would wrap the newly synthesized RNA chain around the DNA double helix. Local opening and unwinding of the DNA solves this problem before it occurs by allowing transcription to proceed on a single face or side of the DNA. In addition, the opening of DNA base pairs during transcription allows Watson–Crick base pairing between template DNA and the bases in the newly synthesized RNA. The process of transcription is divided into three parts: initiation refers to the recognition of an active gene starting point by RNA polymerase and the beginning of the bond formation process; Elongation is the actual synthesis of the RNA chain and is followed by chain termination and release. Page 690 Figure 16.11 Determination of a consensus sequence for prokaryotic promoters. A portion of the data set used for the identification of the consensus sequence for E. coli promoter activity. The –10 region (sometimes called the Pribnow box) is shaded in red and the –35 region nucleotides are colored. Note that none of the individual promoters has the entire consensus sequence. Modified from Rosenberg, M., and Court, D. Ann. Rev. Genet. 13:319, 1979. The Template for RNA Synthesis Is DNA Each cycle of transcription begins and ends with the recognition of specific sites in the DNA template. The DNA sequencing of a large number of transcription start regions, called promoters, has shown that certain conserved sequences occur in promoters with great regularity. An example is shown in Figure 16.11. Similar considerations demonstrate that termination occurs at different conserved sequences. In addition, sites within a transcript may allow premature termination of transcription. These sites can act as molecular switches affecting the continuation of synthesis of an RNA molecule. Conserved sequences near the transcription start are found for both prokaryotic and some eukaryotic promoters. In addition, eukaryotic transcription has been shown in some cases to be affected by internal promoter elements and other sequences called enhancers. Enhancers are genespecific sequences that positively affect transcription. Enhancer sequences can stimulate transcription whether they are located at the beginning, in the middle, or at the end of a gene. An enhancer sequence must be on the same DNA strand as the transcribed gene (genetically in a cis position) but can function in either orientation. Cellular protein factors are known that specifically bind different enhancers. The most likely hypothesis is that protein factors bound to enhancers cause a structural change in the DNA template, allowing protein–protein interaction with other factors or with RNA polymerase itself. This interaction facilitates transcription. RNA Polymerase Catalyzes the Transcription Process RNA polymerases all synthesize RNA in the 5 3 direction using a DNA template; in this respect, they are similar to templatedependent DNA polymerases discussed in Chapter 15. Unlike DNA polymerases, however, RNA polymerases initiate polymerization at a promoter sequence without the need of a DNA Page 691 or RNA primer. Cellular RNA polymerases, both prokaryotic and eukaryotic, are large multisubunit enzymes whose mechanisms are only partially understood. The most intensely studied prokaryotic RNA polymerase is that from Escherichia coli, which consists of five subunits having an aggregate molecular weight of over 500,000 (Table 16.2). Two a subunits, one b subunit, and one subunit constitute the core enzyme, which is capable of faithful transcription but not of specific (i.e., promoterinitiated) RNA synthesis. The addition of a fifth protein subunit, designated s, results in the holoenzyme that is capable of specific RNA synthesis in vitro and in vivo. The logical conclusion, that is involved in the specific recognition of promoters, has been borne out by a variety of biochemical studies and is discussed below. Specific s factors can recognize different classes of genes. For example, a specific factor recognizes promoters for genes that are induced as a result of heat shock. In sporulating bacteria, specific factors recognize genes induced during sporulation. Some bacteriophage synthesize factors that allow the appropriation of the cell's RNA polymerase for transcription of the viral DNA. The common prokaryotic RNA polymerases are inhibited by the antibiotic rifampicin (used in treating tuberculosis), which binds to the b subunit (see Clin. Corr. 16.2). Eukaryotic nuclear RNA polymerases are inhibited differentially by the compound a amanitin, which is synthesized by the poisonous mushroom Amanita phalloides. Three nuclear RNA polymerase classes can be distinguished by these experiments. Very low concentrations of a amanitin inhibit the synthesis of mRNA and some small nuclear RNAs (snRNAs); higher concentrations inhibit the synthesis of tRNA and other snRNAs, whereas rRNA synthesis is not inhibited at these concentrations of drug. Messenger RNA synthesis is the function of RNA polymerase II. Synthesis of transfer RNA, 5sRNA, and some snRNAs are carried out by RNA polymerase III. Ribosomal RNA genes are transcribed by RNA polymerase I, which is concentrated in the nucleolus. (The numbers refer to the order of elution of the enzymes from a chromatography column.) Each enzyme is highly complex structurally (Table 16.2). In addition, a mitochondrial RNA polymerase is responsible for the synthesis of this organelle's mRNA, tRNA, and rRNA species. This enzyme, like bacterial RNA polymerase, is inhibited by rifampicin. TABLE 16.2 Comparative Properties of Some RNA Polymerases Nuclear I (A) II (B) III (C) Mitochondrial E. coli High MW subunitsa 195–197 240–214 155 65 160 ( ) 117–126 140 138 150 ( ) Low MW subunits 61–51 41–34 89 86 ( ) 49–44 29–25 70 40 ( ) 29–25 27–20 53 10 ( ) 19–16.5 19.5 49 19 41 16.5 32 29 19 Variable forms 2–3 types 3–4 types 2–4 types 1 Specialization Nucleolar; rRNA mRNA tRNA All mtRNA Viral RNA 5S rRNA Inhibition by a amanitin Insensitive (>1 mg mL–1) Very sensitive (10–9–10–8 M) Sensitive (10–5–10–4 M) Insensitive, but sensitive to rifampicin Rifampicin sensitive a Molecular weight × 10–3. 1 None Page 692 CLINICAL CORRELATION 16.2 Antibiotics and Toxins That Target RNA Polymerase RNA polymerase is obviously an essential enzyme for life since transcription is the first step of gene expression. No RNA polymerase means no enzymes. Two natural products point out this principle; in both cases inhibition of RNA polymerase leads to death of the organism. The ''death cap" or "destroying angel" mushroom, Amanita phalloides, is highly poisonous and still causes several deaths each year despite widespread warnings to amateur mushroom hunters (it is reputed to taste delicious, incidentally). The most lethal toxin, a amanitin, inhibits the largest subunit of eukaryotic RNA polymerase II, thereby inhibiting mRNA synthesis. The course of the poisoning is twofold: initial, relatively mild, gastrointestinal symptoms are followed about 48 h later by massive liver failure as essential mRNAs and their proteins are degraded but not replaced by newly synthesized molecules. The only therapy is supportive, including liver transplantation; but this latter course is clearly a desperate measure of unproven efficacy. More benign (at least from the point of view of our own species) is the action of the antibiotic rifampicin to inhibit the RNA polymerases of a variety of bacteria, most notably in the treatment of tuberculosis. Mycobacterium tuberculosis, the causative agent, is insensitive to many commonly used antibiotics, but it is sensitive to rifampicin, the product of a soil streptomycetes. Since mammalian RNA polymerase is so different from the prokaryotic variety, inhibition of the latter enzyme is possible without great toxicity to the host. This consideration implies a good therapeutic index for the drug, that is, the ability to treat a disease without causing undue harm to the patient. Together with improved public health measures, antibiotic therapy with rifampicin and isoniazid (an antimetabolite) has greatly reduced the morbidity due to tuberculosis in industrialized countries. Unfortunately, the disease is still endemic in impoverished populations in the United States and in other countries. Furthermore, in increasing numbers, immunocompromised individuals, especially AIDS patients, have active tuberculosis. Mitchel, D. H. Amanita mushroom poisoning. Annu. Rev. Med. 31:51, 1980; Gilman, A. G., Rall, T. W., Nies, A. S., and Taylor, P. (Eds.). The Pharmacological Basis of Therapeutics, 8th ed. New York: Pergamon Press, 1990, pp. 129–130; DeCock, K. M., Soro, B., Colibaly, I. M., Lucas, S. B. Tuberculosis and HIV infection in sub Saharan Africa. JAMA 268:1581, 1992. The Steps of Transcription in Prokaryotes Have Been Determined Transcription is a strandselective process; most double helical DNA is transcribed in only one direction. This is illustrated as follows: The DNA strand that serves as the template for RNA synthesis is sometimes called the sense strand because it is complementary to the RNA transcript. Conventionally, the sense strand is usually the "bottom" strand of a doublestranded DNA as written. The other strand, the "top" strand, has the same direction as the transcript when read in the 5 3 direction; this strand is sometimes (confusingly) called the antisense strand. When only a single DNA sequence is given in this book, the antisense strand is represented. Its sequence can be converted to the RNA transcript of a gene by simply substituting U (uracil) for T (thymine) bases. Prokaryotic transcription begins with the binding of RNA polymerase to a gene's promoter (Figures 16.11 and 16.12). RNA polymerase holoenzyme binds to one face of the DNA extending 45 bp or so upstream and 10 bp downstream from the RNA initiation site. Two short oligonucleotide sequences in this region are highly conserved. One sequence that is located about 10 bp upstream from the transcription start is the consensus sequence (sometimes called a Pribnow box): The positions marked with an asterisk are the most conserved; indeed, the last T residue is always found in E. coli promoters. A second consensus sequence is located upstream from the Pribnow or "–10" box. This "–35 sequence" Page 693 Figure 16.12 Early events in prokaryotic transcription. (a) Recognition: RNA polymerase (drawn smaller than scale) with "sigma" factor binds to a DNA promoter region in a "closed" conformation. (b) Initiation: The complex is converted to an "open" conformation and the first nucleoside triphosphate aligns with the DNA. (c) Bond formation: The first phosphodiester bond is formed and the "sigma'' factor released. (d) Elongation: Synthesis of nascent RNA proceeds with movement of the RNA polymerase along the DNA. The double helix reforms. Page 694 is centered about 35 bp upstream from the transcription start; the nucleotides with asterisks are most conserved. The spacing between the –35 and –10 sequences is crucial with 17 bp being highly conserved. As shown in Figure 16.13, the TTGACA and TATAAT sequences are asymmetrical, that is, they do not have the same sequence if the complementary sequence is read. Thus the promoter sequence itself determines that transcription will proceed in only one direction. What difference do the consensus sequences make to a gene? Measurements of RNA polymerase binding affinity and initiation efficiency to Figure 16.13 Biosynthesis of RNA showing asymmetry in transcription. Nucleoside 5 triphosphates align with complementary bases on one DNA strand, the template. RNA polymerase catalyzes the formation of the 3 ,5 phosphodiester links by attaching the 5 phosphate of the incoming nucleotide to the 3 OH group of the growing nascent RNA releasing P . i The new RNA is synthesized from its 5 end toward the 3 end. Page 695 various promoter sequences have shown that the most active promoters fit the consensus sequences most closely. Statistical measurements of promoter homology conform closely to the measured "strength" of a promoter, that is, its kinetic ability to initiate transcription with –35 purified RNA polymerase. Bases flanking the –35 and –10 sequences, bases near the transcription start, and bases located near the –16 position are weakly conserved. In some of these weakly conserved regions, RNA polymerase may require that a particular nucleotide not be present or that local variations in DNA helical structure be present. Promoters for E. coli heat shock genes have different consensus sequences at the –35 and –10 homologies. This is consistent with their being recognized by a different factor. An RNA transcript usually starts with a purine riboside triphosphate; that is, pppG . . . or pppA . . ., but pyrimidine starts are also known (Figures 16.11 and 16.12). The position of transcription initiation differs slightly among various promoters but usually is from five to eight base pairs downstream from the invariant T of the Pribnow box. Initiation Two kinetically distinct steps are required for RNA polymerase to initiate the synthesis of an RNA transcript. In the first step, RNA polymerase holoenzyme binds to the promoter DNA to form a "closed complex." In the second step, the holoenzyme forms a more tightly bound "open complex," which is characterized by a local opening of about 10 bp of the DNA double helix. Since the consensus Pribnow box is AT rich, it can facilitate this local unwinding. As discussed in Chapter 14, opening 10 bp of DNA is topologically equivalent to the relaxation of a single negative supercoil. As might be predicted from this observation, the activity of some promoters depends on the superhelical state of the DNA template; some promoters are more active on highly supercoiled DNA while others are more active when the superhelical density of the template is lower. The unwound DNA binds the initiating triphosphate and RNA polymerase then forms the first phosphodiester bond. The enzyme translocates to the next position (this is the rifampicininhibited step) and continues synthesis. At or a short time after the initial bond formation, factor is released and the enzyme is considered to be in an elongation mode. Other RNA polymerase molecules can now bind to the promoter so that a gene can be transcribed many times (Figure 16.14). Figure 16.14 Simultaneous transcription of a gene by many RNA polymerases, depicting the increasing length of nascent RNA molecules. Courtesy of Dr. O. L. Miller, University of Virginia. Reproduced with permission from Miller, O. L., and Beatty, B. R. J. Cell Physiol. 74:225, 1969. Elongation RNA polymerase continues the binding–bond formation–translocation cycle at a rate of about 40 nucleotides per second. This rate is only an average, however, and there are many examples known for which RNA polymerase pauses or slows down at particular sequences, usually inverted repeats (palindrome sequence of nucleotides). As will be discussed below, these pauses can bring about transcription termination. As RNA polymerase continues down the double helix, it continues to separate the two strands of the DNA template. As seen in Figure 16.12, this process allows the template (sense) strand of the DNA to base pair with the growing RNA chain. Thus a single mechanism of information transfer (Watson–Crick base pairing) serves several processes: DNA replication, DNA repair, and transcription of genetic information into RNA. (As will be seen in Chapter 17, base pairing is essential for translation as well.) The process of unwinding and restoring the DNA double helix is aided by DNA topoisomerases I and II, which are components of the transcription complex. Changes in the transcription complex during the elongation phase can affect subsequent termination events. These changes depend on the binding of another cellular protein (nusA protein) to core RNA polymerase. Failure to Page 696 Figure 16.15 The stem–loop structure of the RNA transcript that determines rhoindependent transcriptional termination. Note the two components of the structure: the G + Crich stem and loop,followed by a sequence of U residues. bind sometimes results in an increased frequency of termination and, consequently, a reduced level of gene expression. Termination The RNA polymerase complex also recognizes the ends of genes (Figure 16.15). Transcription termination can occur in either of two modes, depending on whether or not it is dependent on the protein factor rho. Terminators are thus classified as rho independent or rho dependent. Rhoindependent terminators are better characterized (Figure 16.15). A consensustype sequence is involved here: a GC rich palindrome (inverted repeat) precedes a sequence of 6–7 U residues in the RNA chain. As a result the RNA chain forms a stem and loop structure preceding the U residues. The secondary structure of the stem and loop is crucial for termination; base change mutations in the stem and loop that disrupt pairing also reduce termination. Furthermore, the most efficient terminators are the most GC rich and therefore most stable. The terminator stem and loop stabilize prokaryotic mRNA against nucleolytic degradation. Rhodependent terminators are less well defined. Rho factor is a hexameric protein possessing an essential RNAdependent ATPase activity. The sequences of rhodependent termination sites feature regularly spaced C residues within a relatively unstructured length of the transcript. The nascent RNA is thought to wrap around rho factor while ATP hydrolysis leads to dissociation of the transcript from the template. Prokaryotic ribosomes usually attach to the nascent mRNA while it is being transcribed. This coupling between transcription and translation is important in gene control by attenuation, which is discussed in Chapter 19. Transcription in Eukaryotes Involves Many Additional Molecular Events Eukaryotic transcription is considerably more complex than the process in prokaryotes. While the information specifying a promoter is still carried in a DNA sequence, several molecular events besides RNA polymerase binding are required for transcription initiation. First, chromatin containing the promoter sequence must be spatially accessible to the transcription machinery. Second, protein transcription factors distinct from RNA polymerase must bind to sequences in the promoter region for a gene to be active. Third, other sequences located some distance away from the promoter affect transcription; these sequences are termed enhancers and they, too, bind protein factors to stimulate transcription. Finally, recall that the eukaryotic RNA polymerase consists of three distinct enzyme forms, each specific form capable of transcribing only a Figure 16.16 DNasehypersensitive (DH) sites upstream of the promoter for the chick lysozyme gene, a typical eukaryotic transcriptional unit. Hypersensitive sites, that is, sequences around the lysozyme gene where nucleosomes are not bound to the DNA, are indicated by arrows. Note that some hypersensitive sites are found in the lysozyme promoter whether the oviduct is synthesizing or not synthesizing lysozyme; the synthesis of lysozyme is accompanied by the opening up of a new hypersensitive site in mature oviduct. In contrast, no hypersensitive sites are present in nucleated erythrocytes that never synthesize lysozyme. Adapted from Elgin, S. C. R. J. Biol. Chem. 263:1925, 1988. Page 697 CLINICAL CORRELATION 16.3 Fragile X Syndrome: A Chromatin Disease? Fragile X syndrome is the single most common form of inherited mental retardation, affecting 1/1250 males and 1/2000 females. A variety of anatomical and neurological symptoms result from the inactivation of the FMR1 gene, located on the X chromosome. The genetics of the syndrome are complex due to the molecular mechanism of the Fragile X mutation. The Fragile X condition results from the expansion of a trinucleotide repeat sequence, CGG, found at the 5 untranslated region of the FRM1 gene. Normally, this repeat is present in 30 copies, although normal individuals can have up to 200 copies of the repeat. In individuals with Fragile X syndrome, the FMR1 gene contains many more copies, from 200 to thousands, of the CGG repeat. The complex genetics of the disease result from the potential of the CGG repeat sequence to expand from generation to generation. The presence of an abnormally high number of CGG repeats induces extensive DNA methylation of the entire promoter region of FMR1. Methylated DNA is transcriptionally inactive, so FMR1 mRNA is not synthesized. The absence of FMR1 protein leads to the pathology of the disease. FMR1 protein normally is located in the cytoplasm in all tissues of the early fetus and, later, especially in the fetal brain. Its sequence has some characteristics of an RNA binding protein. One hypothesis is that the protein aids in the translation of brainspecific mRNAs during development. Warren, S. L., and Nelson, D. L. Advances in molecular analysis of Fragile X syndrome. JAMA 271:536, 1994; and Caskey, C. T. Triple repeat mutations in human disease. Science 256:784, 1992. single class of cellular RNA. By contrast, transcription in prokaryotes requires, in the simplest case, only an appropriate sequence of DNA, RNA polymerase holoenzyme, and nucleoside triphosphate substrates. The Nature of Active Chromatin The structural organization of eukaryotic chromosomes was discussed in Chapter 14. Although chromatin is organized into nucleosomes whether or not it is capable of being transcribed, an active gene has a generally "looser" configuration than does transcriptionally inactive chromatin. This difference is most striking in the promoter sequences, parts of which are not organized into nucleosomes at all (Figure 16.16). The lack of nucleosomes is manifested experimentally by the enhanced sensitivity of promoter sequences to external reagents that cleave DNA, such as the enzyme DNase I. This enhanced accessibility of promoter sequences (termed DNase I hypersensitivity) ensures that transcriptional factors will be able to bind to appropriate regulatory sequences. In addition, although the transcribed parts of a gene may be organized into nucleosomes, the nucleosomes are less tightly bound than those in an inactive gene. Finally, DNA may be transcriptionally inactivated by methylation (see Clin. Corr. 16.3). The overall theme is one of partially unfolded chromatin being necessary but not sufficient for transcription. Enhancers Enhancer sequences increase (enhance) the expression of a gene about 100fold, hence the name. They function only when located on the same DNA molecule (chromosome) as the promoter whose activity they affect. They can function when located in either the 5 or 3 direction and as much as 1000 bp away from the relevant promoter. Protein factors bind to enhancer DNA and are necessary for enhancer function. Transcription of Ribosomal RNA Genes Recall that rRNA genes are located in a specialized nuclear structure, the nucleolus. There are several hundred copies of each rRNA gene in a eukaryotic cell, tandemly repeated in the DNA of a specific region of one chromosome, the nucleolar organizer. The repeat units contain a copy of each RNA sequence (28S, 5.8S, and 18S) and are separated from each other by nontranscribed spacer regions. Figure 16.17 is a diagram of this arrangement. Each repeat unit is transcribed as a unit, yielding a primary transcript containing one copy each of the 28S, 5.8S, and 18S sequences, ensuring synthesis of equimolar amounts of these three RNAs. The primary transcript is then processed by ribonucleases and modifying enzymes to the three mature rRNA species (see Section 16.5). Termination of transcription occurs within the nontranscribed spacer region before RNA polymerase I reaches the promoter of the next repeat unit. The promoter recognized by RNA polymerase I is located within the nontranscribed spacer, from about positions –40 to +10 and from –150 to –110. A transcription factor binds to the promoter and thereby directs RNA polymerase Figure 16.17 Structure of a rRNA transcription unit. Ribosomal RNA genes are arranged with many copies one after another. Each copy is transcribed separately and each transcript is processed into three separate RNA species. Promoter and enhancer sequences are located in the nontranscribed regions of the tandemly repeated sequences. Page 698 recognition of the promoter sequence. In addition, an enhancer element is located about 250 bp upstream from the promoter in human ribosomal DNA. The size of the nontranscribed spacer varies considerably from one organism to the next, as does the position of the enhancer element. Transcription of rRNA can be very rapid; this reflects the fact that synthesis of ribosomes is ratelimiting for cell growth. Phosphorylation of RNA polymerase I may activate especially rapid transcription of rRNA, for example, during embryonic growth or liver regeneration. Transcription by RNA Polymerase II RNA polymerase II is responsible for the synthesis of mRNA in the nucleus. Three common themes have emerged from research on a large number of genes (Figure 16.18). (1) The DNA sequences controlling transcription are complex; a single gene may be controlled by as many as six or eight DNA sequence elements in addition to the promoter (RNA polymerase binding region) itself. The controlling sequence elements function in combination to give a finely tuned pattern of control. (2) The effect of the controlling sequences on transcription is mediated by the binding of protein molecules to each sequence element. These transcription factors recognize the nucleotide sequence of the appropriate controlling sequence element. (3) Bound transcription factors bind with each other and with RNA polymerase to activate transcription. The DNA binding and activation activities of the factors reside in separate domains of the proteins. Figure 16.18 Interaction of transcription factors with promoters. A large number of transcriptional factors interact with eukaryotic promoter regions. (a) A hypothetical array of factors that interact with specific DNA sequences near the promoter. This includes a factor, TFIID, which binds to the TATA box and the Jun and Fos proteins, which are protooncogenes (Clin. Corr. 16.4). The figure is not meant to imply that all of the DNA binding factors bind to the promoter simultaneously. (b) One way in which the DNA binding factors are hypothesized to bind to each other and to RNA polymerase. Although this model is not completely proved, it is known that proteins that bind to distant DNA sequences make protein–protein contacts with each other. Reprinted with permission from Mitchell, P. J., and Tjian, R. Science 245:371, 1989.