مهندسی ژنتیک.بیوتکنولوژی.ژنتیک مولکولی

How Cells Read the Genome: From DNA to Protein

Only when the structure of DNA was discovered in the early 1950s did it become clear how the hereditary information in cells is encoded in DNA's sequence of nucleotides. The progress since then has been astounding. Fifty years later, we have complete genome sequences for many organisms, including humans, and we therefore know the maximum amount of information that is required to produce a complex organism like ourselves. The limits on the hereditary information needed for life constrain the biochemical and structural features of cells and make it clear that biology is not infinitely complex.

In this chapter, we explain how cells decode and use the information in their genomes. We shall see that much has been learned about how the genetic instructions written in an alphabet of just four “letters”—the four different nucleotides in DNA—direct the formation of a bacterium, a fruit fly, or a human. Nevertheless, we still have a great deal to discover about how the information stored in an organism's genome produces even the simplest unicellular bacterium with 500 genes, let alone how it directs the development of a human with approximately 30,000 genes. An enormous amount of ignorance remains; many fascinating challenges therefore await the next generation of cell biologists.

The problems cells face in decoding genomes can be appreciated by considering a small portion of the genome of the fruit fly Drosophila melanogaster (Figure 6-1). Much of the DNA-encoded information present in this and other genomes is used to specify the linear order—the sequence—of amino acids for every protein the organism makes. As described in Chapter 3, the amino acid sequence in turn dictates how each protein folds to give a molecule with a distinctive shape and chemistry. When a particular protein is made by the cell, the corresponding region of the genome must therefore be accurately decoded. Additional information encoded in the DNA of the genome specifies exactly when in the life of an organism and in which cell types each gene is to be expressed into protein. Since proteins are the main constituents of cells, the decoding of the genome determines not only the size, shape, biochemical properties, and behavior of cells, but also the distinctive features of each species on Earth.

One might have predicted that the information present in genomes would be arranged in an orderly fashion, resembling a dictionary or a telephone directory. Although the genomes of some bacteria seem fairly well organized, the genomes of most multicellular organisms, such as our Drosophila example, are surprisingly disorderly. Small bits of coding DNA (that is, DNA that codes for protein) are interspersed with large blocks of seemingly meaningless DNA. Some sections of the genome contain many genes and others lack genes altogether. Proteins that work closely with one another in the cell often have their genes located on different chromosomes, and adjacent genes typically encode proteins that have little to do with each other in the cell. Decoding genomes is therefore no simple matter. Even with the aid of powerful computers, it is still difficult for researchers to locate definitively the beginning and end of genes in the DNA sequences of complex genomes, much less to predict when each gene is expressed in the life of the organism. Although the DNA sequence of the human genome is known, it will probably take at least a decade for humans to identify every gene and determine the precise amino acid sequence of the protein it produces. Yet the cells in our body do this, thousands of times a second.

The DNA in genomes does not direct protein synthesis itself, but instead uses RNA as an intermediary molecule. When the cell needs a particular protein, the nucleotide sequence of the appropriate portion of the immensely long DNA molecule in a chromosome is first copied into RNA (a process called transcription). It is these RNA copies of segments of the DNA that are used directly as templates to direct the synthesis of the protein (a process called translation). The flow of genetic information in cells is therefore from DNA to RNA to protein (Figure 6-2). All cells, from bacteria to humans, express their genetic information in this way—a principle so fundamental that it is termed the central dogma of molecular biology.

Despite the universality of the central dogma, there are important variations in the way information flows from DNA to protein. Principal among these is that RNA transcripts in eucaryotic cells are subject to a series of processing steps in the nucleus, including RNA splicing, before they are permitted to exit from the nucleus and be translated into protein. These processing steps can critically change the “meaning” of an RNA molecule and are therefore crucial for understanding how eukaryotic cells read the genome. Finally, although we focus on the production of the proteins encoded by the genome in this chapter, we see that for some genes RNA is the final product. Like proteins, many of these RNAs fold into precise three-dimensional structures that have structural and catalytic roles in the cell.

We begin this chapter with the first step in decoding a genome: the process of transcription by which an RNA molecule is produced from the DNA of a gene. We then follow the fate of this RNA molecule through the cell, finishing when a correctly folded protein molecule has been formed. At the end of the chapter, we consider how the present, quite complex, scheme of information storage, transcription, and translation might have arisen from simpler systems in the earliest stages of cellular evolution

 Figure 6-1. Schematic depiction of a portion of chromosome 2 from the genome of the fruit fly Drosophila melanogaster . This figure represents approximately 3% of the total Drosophila genome, arranged as six contiguous segments. As summarized in the key, the symbolic representations are: rainbow-colored bar: G-C base-pair content; black vertical lines of various thicknesses: locations of transposable elements, with thicker bars indicating clusters of elements; colored boxes: genes (both known and predicted) coded on one strand of DNA (boxes above the midline) and genes coded on the other strand (boxes below the midline). The length of each predicted gene includes both its exons (protein-coding DNA) and its introns (non-coding DNA) (see Figure 4-25). As indicated in the key, the height of each gene box is proportional to the number of cDNAs in various databases that match the gene. As described in Chapter 8, cDNAs are DNA copies of mRNA molecules, and large collections of the nucleotide sequences of cDNAs have been deposited in a variety of databases. The higher the number of matches between the nucleotide sequences of cDNAs and that of a particular predicted gene, the higher the confidence that the predicted gene is transcribed into RNA and is thus a genuine gene. The color of each gene box (see color code in the key) indicates whether a closely related gene is known to occur in other organisms. For example, MWY means the gene has close relatives in mammals, in the nematode worm Caenorhabditis elegans, and in the yeast Saccharomyces cerevisiae. MW indicates the gene has close relatives in mammals and the worm but not in yeast. (From Mark D. Adams et al., Science 287:2185–2195, 2000. © AAAS.)

 Figure 6-2. The pathway from DNA to protein. The flow of genetic information from DNA to RNA (transcription) and from RNA to protein (translation) occurs in all living cells.

 From DNA to RNA

Transcription and translation are the means by which cells read out, or express, the genetic instructions in their genes. Because many identical RNA copies can be made from the same gene, and each RNA molecule can direct the synthesis of many identical protein molecules, cells can synthesize a large amount of protein rapidly when necessary. But each gene can also be transcribed and translated with a different efficiency, allowing the cell to make vast quantities of some proteins and tiny quantities of others (Figure 6-3). Moreover, as we see in the next chapter, a cell can change (or regulate) the expression of each of its genes according to the needs of the moment—most obviously by controlling the production of its RNA.

 Figure 6-3. Genes can be expressed with different efficiencies. Gene A is transcribed and translated much more efficiently than gene B. This allows the amount of protein A in the cell to be much greater than that of protein B.

 Portions of DNA Sequence Are Transcribed into RNA

The first step a cell takes in reading out a needed part of its genetic instructions is to copy a particular portion of its DNA nucleotide sequence—a gene—into an RNA nucleotide sequence. The information in RNA, although copied into another chemical form, is still written in essentially the same language as it is in DNA—the language of a nucleotide sequence. Hence the name transcription.

Like DNA, RNA is a linear polymer made of four different types of nucleotide subunits linked together by phosphodiester bonds (Figure 6-4). It differs from DNA chemically in two respects: (1) the nucleotides in RNA are ribonucleotides—that is, they contain the sugar ribose (hence the name ribonucleic acid) rather than deoxyribose; (2) although, like DNA, RNA contains the bases adenine (A), guanine (G), and cytosine (C), it contains the base uracil (U) instead of the thymine (T) in DNA. Since U, like T, can base-pair by hydrogen-bonding with A (Figure 6-5), the complementary base-pairing properties described for DNA in Chapters 4 and 5 apply also to RNA (in RNA, G pairs with C, and A pairs with U). It is not uncommon, however, to find other types of base pairs in RNA: for example, G pairing with U occasionally.

 Figure 6-4. The chemical structure of RNA. (A) RNA contains the sugar ribose, which differs from deoxyribose, the sugar used in DNA, by the presence of an additional -OH group. (B) RNA contains the base uracil, which differs from thymine, the equivalent base in DNA, by the absence of a -CH₃ group. (C) A short length of RNA. The phosphodiester chemical linkage between nucleotides in RNA is the same as that in DNA.

 Despite these small chemical differences, DNA and RNA differ quite dramatically in overall structure. Whereas DNA always occurs in cells as a double-stranded helix, RNA is single-stranded. RNA chains therefore fold up into a variety of shapes, just as a polypeptide chain folds up to form the final shape of a protein (Figure 6-6). As we see later in this chapter, the ability to fold into complex three-dimensional shapes allows some RNA molecules to have structural and catalytic functions.

Figure 6-5. Uracil forms base pairs with adenine. The absence of a methyl group in U has no effect on base-pairing; thus, U-A base pairs closely resemble T-A base pairs (see Figure 4-4).

 Figure 6-6. RNA can fold into specific structures. RNA is largely single-stranded, but it often contains short stretches of nucleotides that can form conventional base-pairs with complementary sequences found elsewhere on the same molecule. These interactions, along with additional “nonconventional” base-pair interactions, allow an RNA molecule to fold into a three-dimensional structure that is determined by its sequence of nucleotides. (A) Diagram of a folded RNA structure showing only conventional base-pair interactions; (B) structure with both conventional (red) and nonconventional (green) base-pair interactions; (C) structure of an actual RNA, a portion of a group 1 intron (see Figure 6-36). Each conventional base-pair interaction is indicated by a “rung” in the double helix. Bases in other configurations are indicated by broken rungs.

 Transcription Produces RNA Complementary to One Strand of DNA

All of the RNA in a cell is made by DNA transcription, a process that has certain similarities to the process of DNA replication discussed in Chapter 5. Transcription begins with the opening and unwinding of a small portion of the DNA double helix to expose the bases on each DNA strand. One of the two strands of the DNA double helix then acts as a template for the synthesis of an RNA molecule. As in DNA replication, the nucleotide sequence of the RNA chain is determined by the complementary base-pairing between incoming nucleotides and the DNA template. When a good match is made, the incoming ribonucleotide is covalently linked to the growing RNA chain in an enzymatically catalyzed reaction. The RNA chain produced by transcription—the transcript—is therefore elongated one nucleotide at a time, and it has a nucleotide sequence that is exactly complementary to the strand of DNA used as the template (Figure 6-7).

Figure 6-7. DNA transcription produces a single-stranded RNA molecule that is complementary to one strand of DNA.

Transcription, however, differs from DNA replication in several crucial ways. Unlike a newly formed DNA strand, the RNA strand does not remain hydrogen-bonded to the DNA template strand. Instead, just behind the region where the ribonucleotides are being added, the RNA chain is displaced and the DNA helix re-forms. Thus, the RNA molecules produced by transcription are released from the DNA template as single strands. In addition, because they are copied from only a limited region of the DNA, RNA molecules are much shorter than DNA molecules. A DNA molecule in a human chromosome can be up to 250 million nucleotide-pairs long; in contrast, most RNAs are no more than a few thousand nucleotides long, and many are considerably shorter.

The enzymes that perform transcription are called RNA polymerases. Like the DNA polymerase that catalyzes DNA replication (discussed in Chapter 5), RNA polymerases catalyze the formation of the phosphodiester bonds that link the nucleotides together to form a linear chain. The RNA polymerase moves stepwise along the DNA, unwinding the DNA helix just ahead of the active site for polymerization to expose a new region of the template strand for complementary base-pairing. In this way, the growing RNA chain is extended by one nucleotide at a time in the 5′-to-3′ direction (Figure 6-8). The substrates are nucleoside triphosphates (ATP, CTP, UTP, and GTP); as for DNA replication, a hydrolysis of high-energy bonds provides the energy needed to drive the reaction forward (see Figure 5-4).

 Figure 6-8. DNA is transcribed by the enzyme RNA polymerase. The RNA polymerase (pale blue) moves stepwise along the DNA, unwinding the DNA helix at its active site. As it progresses, the polymerase adds nucleotides (here, small “T” shapes) one by one to the RNA chain at the polymerization site using an exposed DNA strand as a template. The RNA transcript is thus a single-stranded complementary copy of one of the two DNA strands. The polymerase has a rudder (see Figure 6-11) that displaces the newly formed RNA, allowing the two strands of DNA behind the polymerase to rewind. A short region of DNA/RNA helix (approximately nine nucleotides in length) is therefore formed only transiently, and a “window” of DNA/RNA helix therefore moves along the DNA with the polymerase. The incoming nucleotides are in the form of ribonucleoside triphosphates (ATP, UTP, CTP, and GTP), and the energy stored in their phosphate-phosphate bonds provides the driving force for the polymerization reaction (see Figure 5-4). (Adapted from a figure kindly supplied by Robert Landick.)

The almost immediate release of the RNA strand from the DNA as it is synthesized means that many RNA copies can be made from the same gene in a relatively short time, the synthesis of additional RNA molecules being started before the first RNA is completed (Figure 6-9). When RNA polymerase molecules follow hard on each other's heels in this way, each moving at about 20 nucleotides per second (the speed in eukaryotes), over a thousand transcripts can be synthesized in an hour from a single gene.

 Figure 6-9. Transcription of two genes as observed under the electron microscope. The micrograph shows many molecules of RNA polymerase simultaneously transcribing each of two adjacent genes. Molecules of RNA polymerase are visible as a series of dots along the DNA with the newly synthesized transcripts (fine threads) attached to them. The RNA molecules (ribosomal RNAs) shown in this example are not translated into protein but are instead used directly as components of ribosomes, the machines on which translation takes place. The particles at the 5′ end (the free end) of each rRNA transcript are believed to reflect the beginnings of ribosome assembly. From the lengths of the newly synthesized transcripts, it can be deduced that the RNA polymerase molecules are transcribing from left to right. (Courtesy of Ulrich Scheer.)

 Although RNA polymerase catalyzes essentially the same chemical reaction as DNA polymerase, there are some important differences between the two enzymes. First, and most obvious, RNA polymerase catalyzes the linkage of ribonucleotides, not deoxyribonucleotides. Second, unlike the DNA polymerases involved in DNA replication, RNA polymerases can start an RNA chain without a primer. This difference may exist because transcription need not be as accurate as DNA replication (see Table 5-1, p. 243). Unlike DNA, RNA does not permanently store genetic information in cells. RNA polymerases make about one mistake for every 10⁴ nucleotides copied into RNA (compared with an error rate for direct copying by DNA polymerase of about one in 10⁷ nucleotides), and the consequences of an error in RNA transcription are much less significant than that in DNA replication.

Although RNA polymerases are not nearly as accurate as the DNA polymerases that replicate DNA, they nonetheless have a modest proofreading mechanism. If the incorrect ribonucleotide is added to the growing RNA chain, the polymerase can back up, and the active site of the enzyme can perform an excision reaction that mimics the reverse of the polymerization reaction, except that water instead of pyrophosphate is used (see Figure 5-4). RNA polymerase hovers around a misincorporated ribonucleotide longer than it does for a correct addition, causing excision to be favored for incorrect nucleotides. However, RNA polymerase also excises many correct bases as part of the cost for improved accuracy.

 Cells Produce Several Types of RNA

The majority of genes carried in a cell's DNA specify the amino acid sequence of proteins; the RNA molecules that are copied from these genes (which ultimately direct the synthesis of proteins) are called messenger RNA (mRNA) molecules. The final product of a minority of genes, however, is the RNA itself. Careful analysis of the complete DNA sequence of the genome of the yeast S. cerevisiae has uncovered well over 750 genes (somewhat more than 10% of the total number of yeast genes) that produce RNA as their final product, although this number includes multiple copies of some highly repeated genes. These RNAs, like proteins, serve as enzymatic and structural components for a wide variety of processes in the cell. In Chapter 5 we encountered one of those RNAs, the template carried by the enzyme telomerase. Although not all of their functions are known, we see in this chapter that some small nuclear RNA (snRNA) molecules direct the splicing of pre-mRNA to form mRNA, that ribosomal RNA (rRNA) molecules form the core of ribosomes, and that transfer RNA (tRNA) molecules form the adaptors that select amino acids and hold them in place on a ribosome for incorporation into protein (Table 6-1).

Table 6-1. Principal Types of RNAs Produced in Cells

Each transcribed segment of DNA is called a transcription unit. In eukaryotes, a transcription unit typically carries the information of just one gene, and therefore codes for either a single RNA molecule or a single protein (or group of related proteins if the initial RNA transcript is spliced in more than one way to produce different mRNAs). In bacteria, a set of adjacent genes is often trans-cribed as a unit; the resulting mRNA molecule therefore carries the information for several distinct proteins.

Overall, RNA makes up a few percent of a cell's dry weight. Most of the RNA in cells is rRNA; mRNA comprises only 3–5% of the total RNA in a typical mammalian cell. The mRNA population is made up of tens of thousands of different species, and there are on average only 10–15 molecules of each species of mRNA present in each cell.

 Signals Encoded in DNA Tell RNA  Polymerase Where to Start and Stop

To transcribe a gene accurately, RNA polymerase must recognize where on the genome to start and where to finish. The way in which RNA polymerases perform these tasks differs somewhat between bacteria and eukaryotes. Because the process in bacteria is simpler, we look there first.

The initiation of transcription is an especially important step in gene expression because it is the main point at which the cell regulates which proteins are to be produced and at what rate. Bacterial RNA polymerase is a multisubunit complex. A detachable subunit, called sigma (σ) factor, is largely responsible for its ability to read the signals in the DNA that tell it where to begin transcribing (Figure 6-10). RNA polymerase molecules adhere only weakly to the bacterial DNA when they collide with it, and a polymerase molecule typically slides rapidly along the long DNA molecule until it dissociates again. However, when the polymerase slides into a region on the DNA double helix called a promoter, a special sequence of nucleotides indicating the starting point for RNA synthesis, it binds tightly to it. The polymerase, using its σ factor, recognizes this DNA sequence by making specific contacts with the portions of the bases that are exposed on the outside of the helix (Step 1 in Figure 6-10).

After the RNA polymerase binds tightly to the promoter DNA in this way, it opens up the double helix to expose a short stretch of nucleotides on each strand (Step 2 in Figure 6-10). Unlike a DNA helicase reaction (see Figure 5-15), this limited opening of the helix does not require the energy of ATP hydrolysis. Instead, the polymerase and DNA both undergo reversible structural changes that result in a more energetically favorable state. With the DNA unwound, one of the two exposed DNA strands acts as a template for complementary base-pairing with incoming ribonucleotides (see Figure 6-7), two of which are joined together by the polymerase to begin an RNA chain. After the first ten or so nucleotides of RNA have been synthesized (a relatively inefficient process during which polymerase synthesizes and discards short nucleotide oligomers), the σ factor relaxes its tight hold on the polymerase and eventually dissociates from it. During this process, the polymerase undergoes additional structural changes that enable it to move forward rapidly, transcribing without the σ factor (Step 4 in Figure 6-10). Chain elongation continues (at a speed of approximately 50 nucleotides/sec for bacterial RNA polymerases) until the enzyme encounters a second signal in the DNA, the terminator (described below), where the polymerase halts and releases both the DNA template and the newly made RNA chain (Step 7 in Figure 6-10). After the polymerase has been released at a terminator, it reassociates with a free σ factor and searches for a new promoter, where it can begin the process of transcription again.

  Figure 6-10. The transcription cycle of bacterial RNA polymerase. In step 1, the RNA polymerase holoenzyme (core polymerase plus σ factor) forms and then locates a promoter (see Figure 6-12). The polymerase unwinds the DNA at the position at which transcription is to begin (step 2) and begins transcribing (step 3). This initial RNA synthesis (sometimes called “abortive initiation”) is relatively inefficient. However, once RNA polymerase has managed to synthesize about 10 nucleotides of RNA, σ relaxes its grip, and the polymerase undergoes a series of conformational changes (which probably includes a tightening of its jaws and the placement of RNA in the exit channel [see Figure 6-11]). The polymerase now shifts to the elongation mode of RNA synthesis (step 4), moving rightwards along the DNA in this diagram. During the elongation mode (step 5) transcription is highly processive, with the polymerase leaving the DNA template and releasing the newly transcribed RNA only when it encounters a termination signal (step 6). Termination signals are encoded in DNA and many function by forming an RNA structure that destabilizes the polymerase's hold on the RNA, as shown here. In bacteria, all RNA molecules are synthesized by a single type of RNA polymerase and the cycle depicted in the figure therefore applies to the production of mRNAs as well as structural and catalytic RNAs. (Adapted from a figure kindly supplied by Robert Landick.)

 Several structural features of bacterial RNA polymerase make it particularly adept at performing the transcription cycle just described. Once the σ factor positions the polymerase on the promoter and the template DNA has been unwound and pushed to the active site, a pair of moveable jaws is thought to clamp onto the DNA (Figure 6-11). When the first 10 nucleotides have been transcribed, the dissociation of σ allows a flap at the back of the polymerase to close to form an exit tunnel through which the newly made RNA leaves the enzyme. With the polymerase now functioning in its elongation mode, a rudder-like structure in the enzyme continuously pries apart the DNA-RNA hybrid formed. We can view the series of conformational changes that takes place during transcription initiation as a successive tightening of the enzyme around the DNA and RNA to ensure that it does not dissociate before it has finished transcribing a gene. If an RNA polymerase does dissociate prematurely, it cannot resume synthesis but must start over again at the promoter.

How do the signals in the DNA (termination signals) stop the elongating polymerase? For most bacterial genes a termination signal consists of a string of A-T nucleotide pairs preceded by a two-fold symmetric DNA sequence, which, when transcribed into RNA, folds into a “hairpin” structure through Watson-Crick base-pairing (see Figure 6-10). As the polymerase transcribes across a terminator, the hairpin may help to wedge open the movable flap on the RNA polymerase and release the RNA transcript from the exit tunnel. At the same time, the DNA-RNA hybrid in the active site, which is held together predominantly by U-A base pairs (which are less stable than G-C base pairs because they form two rather than three hydrogen bonds per base pair), is not sufficiently strong enough to hold the RNA in place, and it dissociates causing the release of the polymerase from the DNA, perhaps by forcing open its jaws. Thus, in some respects, transcription termination seems to involve a reversal of the structural transitions that happen during initiation. The process of termination also is an example of a common theme in this chapter: the ability of RNA to fold into specific structures figures prominently in many aspects of decoding the genome.

Figure 6-11. The structure of a bacterial RNA polymerase. Two depictions of the three-dimensional structure of a bacterial RNA polymerase, with the DNA and RNA modeled in. This RNA polymerase is formed from four different subunits, indicated by different colors (right). The DNA strand used as a template is red, and the non-template strand is yellow. The rudder wedges apart the DNA-RNA hybrid as the polymerase moves. For simplicity only the polypeptide backbone of the rudder is shown in the right-hand figure, and the DNA exiting from the polymerase has been omitted. Because the RNA polymerase is depicted in the elongation mode, the σ factor is absent. (Courtesy of Seth Darst.)

Transcription Start and Stop Signals Are Heterogeneous in Nucleotide Sequence

As we have just seen, the processes of transcription initiation and termination involve a complicated series of structural transitions in protein, DNA, and RNA molecules. It is perhaps not surprising that the signals encoded in DNA that specify these transitions are difficult for researchers to recognize. Indeed, a comparison of many different bacterial promoters reveals that they are heterogeneous in DNA sequence. Nevertheless, they all contain related sequences, reflecting in part aspects of the DNA that are recognized directly by the σ factor. These common features are often summarized in the form of a consensus sequence (Figure 6-12). In general, a consensus nucleotide sequence is derived by comparing many sequences with the same basic function and tallying up the most common nucleotide found at each position. It therefore serves as a summary or “average” of a large number of individual nucleotide sequences.

One reason that individual bacterial promoters differ in DNA sequence is that the precise sequence determines the strength (or number of initiation events per unit time) of the promoter. Evolutionary processes have thus fine-tuned each promoter to initiate as often as necessary and have created a wide spectrum of promoters. Promoters for genes that code for abundant proteins are much stronger than those associated with genes that encode rare proteins, and their nucleotide sequences are responsible for these differences.

Like bacterial promoters, transcription terminators also include a wide range of sequences, with the potential to form a simple RNA structure being the most important common feature. Since an almost unlimited number of nucleotide sequences have this potential, terminator sequences are much more heterogeneous than those of promoters.

We have discussed bacterial promoters and terminators in some detail to illustrate an important point regarding the analysis of genome sequences. Although we know a great deal about bacterial promoters and terminators and can develop consensus sequences that summarize their most salient features, their variation in nucleotide sequence makes it difficult for researchers (even when aided by powerful computers) to definitively locate them simply by inspection of the nucleotide sequence of a genome. When we encounter analogous types of sequences in eukaryotes, the problem of locating them is even more difficult. Often, additional information, some of it from direct experimentation, is needed to accurately locate the short DNA signals contained in genomes.

Promoter sequences are asymmetric (see Figure 6-12), and this feature has important consequences for their arrangement in genomes. Since DNA is double-stranded, two different RNA molecules could in principle be transcribed from any gene, using each of the two DNA strands as a template. However a gene typically has only a single promoter, and because the nucleotide sequences of bacterial (as well as eukaryotic) promoters are asymmetric the polymerase can bind in only one orientation. The polymerase thus has no option but to transcribe the one DNA strand, since it can synthesize RNA only in the 5′ to 3′ direction (Figure 6-13). The choice of template strand for each gene is therefore determined by the location and orientation of the promoter. Genome sequences reveal that the DNA strand used as the template for RNA synthesis varies from gene to gene (Figure 6-14; see also Figure 1-31).

Having considered transcription in bacteria, we now turn to the situation in eucaryotes, where the synthesis of RNA molecules is a much more elaborate affair.

 Figure 6-12. Consensus sequence for the major class of E. coli promoters. (A) The promoters are characterized by two hexameric DNA sequences, the -35 sequence and the -10 sequence named for their approximate location relative to the start point of transcription (designated +1). For convenience, the nucleotide sequence of a single strand of DNA is shown; in reality the RNA polymerase recognizes the promoter as double-stranded DNA. On the basis of a comparison of 300 promoters, the frequencies of the four nucleotides at each position in the -35 and -10 hexamers are given. The consensus sequence, shown below the graph, reflects the most common nucleotide found at each position in the collection of promoters. The sequence of nucleotides between the -35 and -10 hexamers shows no significant similarities among promoters. (B) The distribution of spacing between the -35 and -10 hexamers found in E. coli promoters. The information displayed in these two graphs applies to E. coli promoters that are recognized by RNA polymerase and the major σ factor (designated σ⁷⁰). As we shall see in the next chapter, bacteria also contain minor σ factors, each of which recognizes a different promoter sequence. Some particularly strong promoters recognized by RNA polymerase and σ⁷⁰ have an additional sequence, located upstream (to the left, in the figure) of the -35 hexamer, which is recognized by another subunit of RNA polymerase.

 Figure 6-13. The importance of RNA polymerase orientation. The DNA strand serving as template must be traversed in a 3′ to 5′ direction, as illustrated in Figure 6-9. Thus, the direction of RNA polymerase movement determines which of the two DNA strands is to serve as a template for the synthesis of RNA, as shown in (A) and (B). Polymerase direction is, in turn, determined by the orientation of the promoter sequence, the site at which the RNA polymerase begins transcription.

 Figure 6-14.Directions of transcription along a short portion of a bacterial chromosome. Some genes are transcribed using one DNA strand as a template, while others are transcribed using the other DNA strand. The direction of transcription is determined by the promoter at the beginning of each gene (green arrowheads). Approximately 0.2% (9000 base pairs) of the E. coli chromosome is depicted here. The genes transcribed from left to right use the bottom DNA strand as the template; those transcribed from right to left use the top strand as the template.

 Transcription Initiation in Eukaryotes Requires Many Proteins

In contrast to bacteria, which contain a single type of RNA polymerase, eukaryotic nuclei have three, called RNA polymerase I, RNA polymerase II, and RNA polymerase III. The three polymerases are structurally similar to one another (and to the bacterial enzyme). They share some common subunits and many structural features, but they transcribe different types of genes (Table 6-2). RNA polymerases I and III transcribe the genes encoding transfer RNA, ribosomal RNA, and various small RNAs. RNA polymerase II transcribes the vast majority of genes, including all those that encode proteins, and our subsequent discussion therefore focuses on this enzyme.

Table 6-2. The Three RNA Polymerases in Eucaryotic Cells

Although eucaryotic RNA polymerase II has many structural similarities to bacterial RNA polymerase (Figure 6-15), there are several important differences in the way in which the bacterial and eukaryotic enzymes function, two of which concern us immediately.

 Figure 6-15. Structural similarity between a bacterial RNA polymerase and a eucaryotic RNA polymerase II. Regions of the two RNA polymerases that have similar structures are indicated in green. The eukaryotic polymerase is larger than the bacterial enzyme (12 subunits instead of 5), and some of the additional regions are shown in gray. The blue spheres represent Zn atoms that serve as structural components of the polymerases, and the red sphere represents the Mg atom present at the active site, where polymerization takes place. The RNA polymerases in all modern-day cells (bacteria, archaea, and eucaryotes) are closely related, indicating that the basic features of the enzyme were in place before the divergence of the three major branches of life. (Courtesy of P. Cramer and R. Kornberg.)

 RNA Polymerase II Requires General Transcription Factors

The discovery that, unlike bacterial RNA polymerase, purified eukaryotic RNA polymerase II could not initiate transcription in vitro led to the discovery and purification of the additional factors required for this process. These general transcription factors :

1. help to position the RNA polymerase correctly at the promoter,
2. aid in pulling apart the two strands of DNA to allow transcription to begin,
3. and release RNA polymerase from the promoter into the elongation mode once transcription has begun.

The proteins are “general” because they assemble on all promoters used by RNA polymerase II; consisting of a set of interacting proteins, they are designated as TFII (for transcription factor for polymerase II), and listed as TFIIA, TFIIB, and so on. In a broad sense, the eukaryotic general transcription factors carry out functions equivalent to those of the σ factor in bacteria. Figure 6-16 shows how the general transcription factors assemble in vitro at promoters used by RNA polymerase II.

The assembly process starts with the binding of the general transcription factor TFIID to a short double-helical DNA sequence primarily composed of T and A nucleotides. For this reason, this sequence is known as the TATA sequence, or TATA box, and the subunit of TFIID that recognizes it is called TBP (for TATA-binding protein). The TATA box is typically located 25 nucleotides upstream from the transcription start site. It is not the only DNA sequence that signals the start of transcription (Figure 6-17), but for most polymerase II promoters, it is the most important. The binding of TFIID causes a large distortion in the DNA of the TATA box (Figure 6-18). This distortion is thought to serve as a physical landmark for the location of an active promoter in the midst of a very large genome, and it brings DNA sequences on both sides of the distortion together to allow for subsequent protein assembly steps. Other factors are then assembled, along with RNA polymerase II, to form a complete transcription initiation complex (see Figure 6-16).

After RNA polymerase II has been guided onto the promoter DNA to form a transcription initiation complex, it must gain access to the template strand at the transcription start point. This step is aided by one of the general transcription factors, TFIIH, which contains a DNA helicase. Next, like the bacterial polymerase, polymerase II remains at the promoter, synthesizing short lengths of RNA until it undergoes a conformational change and is released to begin transcribing a gene. A key step in this release is the addition of phosphate groups to the “tail” of the RNA polymerase (known as the CTD or C-terminal domain). This phosphorylation is also catalyzed by TFIIH, which, in addition to a helicase, contains a protein kinase as one of its subunits (see Figure 6-16, D and E). The polymerase can then disengage from the cluster of general transcription factors, undergoing a series of conformational changes that tighten its interaction with DNA and acquiring new proteins that allow it to transcribe for long distances without dissociating.

Once the polymerase II has begun elongating the RNA transcript, most of the general transcription factors are released from the DNA so that they are available to initiate another round of transcription with a new RNA polymerase molecule. As we see shortly, the phosphorylation of the tail of RNA polymerase II also causes components of the RNA processing machinery to load onto the polymerase and thus be in position to modify the newly transcribed RNA as it emerges from the polymerase.

Figure 6-16. Initiation of transcription of a eukaryotic gene by RNA polymerase II. To begin transcription, RNA polymerase requires a number of general transcription factors (called TFIIA, TFIIB, and so on). (A) The promoter contains a DNA sequence called the TATA box, which is located 25 nucleotides away from the site at which transcription is initiated. (B) The TATA box is recognized and bound by transcription factor TFIID, which then enables the adjacent binding of TFIIB (C). For simplicity the DNA distortion produced by the binding of TFIID (see Figure 6-18) is not shown. (D) The rest of the general transcription factors, as well as the RNA polymerase itself, assemble at the promoter. (E) TFIIH then uses ATP to pry apart the DNA double helix at the transcription start point, allowing transcription to begin. TFIIH also phosphorylates RNA polymerase II, changing its conformation so that the polymerase is released from the general factors and can begin the elongation phase of transcription. As shown, the site of phosphorylation is a long C-terminal polypeptide tail that extends from the polymerase molecule. The assembly scheme shown in the figure was deduced from experiments performed in vitro, and the exact order in which the general transcription factors assemble on promoters in cells is not known with certainty. In some cases, the general factors are thought to first assemble with the polymerase, with the whole assembly subsequently binding to the DNA in a single step. The general transcription factors have been highly conserved in evolution; some of those from human cells can be replaced in biochemical experiments by the corresponding factors from simple yeasts.

Figure 6-17. Consensus sequences found in the vicinity of eucaryotic RNA polymerase II start points. The name given to each consensus sequence (first column) and the general transcription factor that recognizes it (last column) are indicated. N indicates any nucleotide, and two nucleotides separated by a slash indicate an equal probability of either nucleotide at the indicated position. In reality, each consensus sequence is a shorthand representation of a histogram similar to that of Figure 6-12. For most RNA polymerase II transcription start points, only two or three of the four sequences are present. For example, most polymerase II promoters have a TATA box sequence, and those that do not typically have a “strong” INR sequence. Although most of the DNA sequences that influence transcription initiation are located “upstream” of the transcription start point, a few, such as the DPE shown in the figure, are located in the transcribed region.

 Figure 6-18. Three-dimensional structure of TBP (TATA-binding protein) bound to DNA. The TBP is the subunit of the general transcription factor TFIID that is responsible for recognizing and binding to the TATA box sequence in the DNA (red). The unique DNA bending caused by TBP—two kinks in the double helix separated by partly unwound DNA—may serve as a landmark that helps to attract the other general transcription factors. TBP is a single polypeptide chain that is folded into two very similar domains (blue and green). (Adapted from J.L. Kim et al., Nature 365:520–527, 1993.)