Transcription in cells and viruses is catalyzed by specialized enzymes called RNA polymerases. The transcription reaction resembles DNA replication in its fundamental chemical mechanism, in the direction of synthesis (5′→3′), and in the requirement for a template strand. And, like replication, transcription has initiation, elongation, and termination phases. In contrast to replication, however, transcription does not require a primer to begin RNA synthesis, and it involves defined sections of DNA rather than the entire molecule. Also, just one of the two strands of a DNA segment serves as the template for a given transcription reaction (Figure 15-1). In this section we discuss the different types of polymerases, steps in the transcription process, and how transcription can be blocked by inhibitors.

RNA polymerases were first discovered by testing cell extracts for activity that could form an RNA polymer from ribonucleoside 5′-triphosphates (rNTPs). Experiments demonstrated that the RNA product of this polymerase activity was complementary to the sequence of DNA supplied in the reaction mix. A particularly telling experiment involved a DNA strand with an alternating (AT)_n sequence. The use of different radiolabeled rNTPs revealed that only ATP and UTP were needed for complete synthesis of RNA, and not GTP or CTP (Figure 15-2). Subsequent experiments using the purified E. coli RNA polymerase, and, later, using bacteriophage RNA polymerases, helped define the fundamental properties of transcription.

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In addition to a DNA template, DNA-dependent RNA polymerases require Mg²⁺ and all four rNTPs (ATP, GTP, UTP, and CTP) as substrates for the polymerization reaction. The chemistry and mechanism of RNA synthesis closely resemble those of DNA synthesis (Figure 15-3). RNA polymerase extends an RNA strand by adding ribonucleotide units to the 3′-hydroxyl end, building RNA in the 5′→3′ direction. The 3′-hydroxyl group makes a nucleophilic attack on the α phosphate of the incoming rNTP, with the concomitant release of pyrophosphate. As noted above, only one of the two DNA strands serves as template. The template DNA is copied in the 3′→5′ direction (antiparallel to the new RNA strand), just as in DNA replication. Each nucleotide in the newly formed RNA is selected by Watson-Crick base pairing: U residues—not T residues, as in DNA—are inserted in the RNA to pair with A residues in the DNA template, G residues are inserted to pair with C residues, and so on (see Figure 6-11). Base-pair geometry may also play a role in nucleotide selection and the resulting fidelity of the polymerase reaction.

RNA polymerases are fascinating enzymes that continue to be actively studied. The simplest examples consist of one polypeptide chain, such as the phage T7 and Sp6 RNA polymerases. In contrast, all cellular RNA polymerases, from bacteria to humans, are composed of multiple polypeptides that fold together to create the functional enzyme. In E. coli, for example, the RNA polymerase core is a large, complex enzyme with five polypeptide subunits: two copies of the α subunit and one copy each of the β, β′, and ω subunits: α₂ββ′ω (M_r 390,000), as shown in Figure 15-4a. A sixth subunit, designated σ and known as sigma factor, binds transiently to the core and directs the enzyme to specific binding sites on the DNA. These six subunits constitute the RNA polymerase holoenzyme. Bacteria have multiple sigma factors, named according to their molecular weight; the most common is σ⁷⁰ (M_r 70,000). Thus, the RNA polymerase holoenzyme of E. coli exists in several forms, depending on the type of σ subunit it contains. Sigma factors play an important role in the recognition of different types of bacterial genes (see Section 15.2).

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In eukaryotic cells, three distinct RNA polymerases are responsible for transcribing RNAs with different functions. RNA polymerase I (Pol I) transcribes genes encoding large rRNA precursors. RNA polymerase II (Pol II) transcribes nearly all protein-coding genes to make mRNA. RNA polymerase III (Pol III) transcribes genes encoding smaller functional RNAs, including tRNAs, some small nuclear RNAs (snRNAs), and 5S ribosomal RNA (the naming of rRNAs is explained in Chapter 18). These enzymes are related to bacterial RNA polymerase at the level of both sequence and structure, indicating that RNA polymerase is an ancient enzyme. However, the eukaryotic RNA polymerases are larger and contain additional proteins not found in bacteria (Table 15-1).

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The molecular structures of bacterial and yeast RNA polymerases have been determined by x-ray crystallography (Figure 15-4b, c). The cleft between the two pincers of the claw contains the enzyme active site and binds two Mg²⁺ ions that facilitate RNA polymerization. The more conserved parts of the polymerase complex are in the interior, whereas regions that have varied more over the course of evolution are at the exterior of the complex.

Unlike DNA polymerase, RNA polymerase does not require a primer to initiate synthesis. RNA polymerase catalyzes RNA synthesis in three distinct phases, similar to those of the DNA polymerase reaction. Initiation occurs as RNA polymerase binds to specific DNA sequences called promoters. Elongation is the process of adding nucleotides to the growing RNA strand. Termination is the release of the product RNA when the polymerase reaches the end of a gene or other transcription unit.

The two strands of a DNA duplex have different roles in transcription. The template strand serves as a template for RNA synthesis, and its complement, the nontemplate strand, is identical in base sequence to the RNA transcribed from the gene, with U in the RNA in place of T in the DNA (Figure 15-5). The nontemplate strand is more often called the coding strand. The coding strand for a particular gene may be located in either strand of a given chromosome.

To enable RNA polymerase to synthesize an RNA strand complementary to the template DNA strand, the DNA duplex must unwind over a short distance, forming what is known as a transcription “bubble” (Figure 15-6). During transcription, the E. coli RNA polymerase generally keeps about 17 bp of DNA unwound. In the elongation phase, the growing end of the new RNA strand base-pairs temporarily with the DNA template in the unwound region to form a short hybrid RNA-DNA double helix, 8 bp in length. The RNA in this hybrid duplex is displaced shortly after its formation as the DNA double helix re-forms. Elongation of a transcript by E. coli RNA polymerase proceeds at a rate of 50 to 90 nucleotides per second. Because DNA is a helix, the movement of a transcription bubble requires considerable strand rotation. Consequently, a moving RNA polymerase generates waves of positive supercoils ahead of the transcription bubble and negative supercoils behind. This has been observed both in the laboratory with purified polymerase enzymes and in live bacterial cells. In the cell, the topological problems caused by transcription are relieved through the action of topoisomerases (see Chapter 9).

RNA polymerases lack a separate proofreading 3′→5′ exonuclease active site, which exists in many DNA polymerases. Consequently, the error rate for transcription is higher than that for chromosomal DNA replication—approximately one error for every 10⁴ to 10⁵ ribonucleotides incorporated into RNA. Because many copies of a transcript are generally produced from a single gene, and all of these are eventually degraded and replaced, a mistake in an RNA molecule is less consequential to the cell than a mistake in the permanent information stored in DNA. Many RNA polymerases, including bacterial RNA polymerase and the eukaryotic Pol II, pause when a mispaired base is added during transcription. In addition, they can remove mismatched nucleotides from the 3′ end of a transcript by direct reversal of the polymerization reaction. However, it is still unclear whether this activity is a true proofreading function and to what extent it may contribute to the fidelity of transcription.

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The general steps of the transcription pathway are the same in both bacteria and eukaryotes (Figure 15-7). The polymerase binds the promoter (step 1), forming first a closed complex, in which the bound DNA is intact, and then an open complex (step 2), in which the bound DNA is partially unwound near a region 10 bp ahead of (upstream of) the transcription start site. Transcription is initiated within the complex (step 3), leading to a conformational change that converts the complex to the form required for elongation. Promoter clearance, involving movement of the transcription complex down the DNA template and away from the promoter, leads to the formation of a tightly bound elongation complex (step 4). Once elongation begins, RNA polymerase becomes a highly efficient enzyme, completing synthesis of the transcript before dissociating from the DNA template (step 5), then recycling for a new round of transcription. Although the steps in this pathway are conserved across species, the details of the process are somewhat more complex in eukaryotic cells.

RNA synthesis is processive, which means that once RNA polymerase begins elongating a transcript, the kinetics of the polymerization reaction greatly favor the addition of the next nucleotide over premature release of the transcript. As we will see, elongation is not a uniform process but instead occurs in fits and starts, and specific sequences trigger termination of RNA synthesis by RNA polymerase.

Small molecules and peptides that inhibit transcription are useful both as antibiotics and as tools for research. Actinomycin D, one of a class of peptide antibiotics isolated from Streptomyces soil bacteria, inhibits transcription elongation by RNA polymerase in bacteria and eukaryotes (Figure 15-8). The planar portion of this molecule intercalates into the double-helical DNA between successive G≡C base pairs, deforming the DNA and preventing movement of the polymerase along the template. Because actinomycin D inhibits RNA elongation both in intact cells and in cell extracts, it is used in the laboratory to identify cell processes that depend on RNA synthesis. Acridine inhibits RNA synthesis in a similar fashion. Rifampicin, a small molecule isolated from Streptomyces mediterranei, inhibits bacterial RNA synthesis by binding to the β subunit of bacterial RNA polymerase, preventing promoter clearance. Because it does not affect the function of eukaryotic polymerases, rifampicin is sometimes used as an antibiotic to treat such bacterial diseases as tuberculosis and leprosy.

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Some species rely on transcription inhibitors for natural biodefense. For example, the mushroom Amanita phalloides produces α-amanitin (Figure 15-9a, b), a cyclic peptide that disrupts eukaryotic mRNA synthesis by blocking Pol II and, at higher concentrations, Pol III. The binding position of α-amanitin in Pol II prevents the flexibility required for translocation of the polymerase along the DNA substrate (Figure 15-9c). α-Amanitin is useful in the laboratory as a specific inhibitor of eukaryotic Pol II, or to determine the polymerase responsible for transcribing a particular gene. This can be demonstrated in an experiment monitoring RNA synthesis when an RNA polymerase and rNTPs are combined with a DNA template (Figure 15-9d). Under normal conditions, Pol I produces rRNA, Pol II produces mRNA, and Pol III produces tRNA. The addition of α-amanitin inhibits the synthesis of mRNA, but not that of rRNA or tRNA. Indeed, Pol I, Pol III and bacterial RNA polymerase are insensitive to α-amanitin—as is the RNA polymerase II of A. phalloides itself!

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Transcription is the first step in the complicated and energy-intensive pathway of protein synthesis, so much of the regulation of protein levels in both bacterial and eukaryotic cells occurs during transcription, particularly its early stages. Because requirements for any gene product vary according to cellular conditions or developmental stage, cells and viruses control transcription so that gene products are made only when they are needed, and in the required proportions. Regulation can occur at any step in transcription, but much of it is directed at the promoter-binding and initiation steps.

The DNA sequence in the promoter region affects the efficiency of RNA polymerase binding and the initiation of transcription. However, differences in promoter sequences are just one of several levels of control during initiation. The binding of additional proteins to sequences both near to and distant from the promoter can also affect transcription levels. Protein binding can activate transcription by facilitating RNA polymerase binding or later steps in the initiation process, or it can repress transcription by blocking polymerase activity (activation and repression of transcription by specific proteins are discussed in detail in Chapters 20 and 21).