A Template DNA Strand Is Transcribed into a Complementary RNA Chain by RNA Polymerase

During transcription of DNA, one DNA strand acts as a template, determining the order in which ribonucleoside triphosphate (rNTP) monomers are linked together to form a complementary RNA chain. Bases in the template DNA strand base-pair with complementary incoming rNTPs, which are then joined in a polymerization reaction catalyzed by RNA polymerase. The polymerization reaction involves a nucleophilic attack by the 3′ oxygen in the growing RNA chain on the α phosphate of the next nucleotide precursor to be added, which results in the formation of a phosphodiester bond and the release of pyrophosphate (PPi). As a consequence of this mechanism, RNA molecules are always synthesized in the 5′→3′ direction (Figure 5-10a).

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FIGURE 5-10 RNA is synthesized 5′→3. (a) Polymerization of ribonucleotides by RNA polymerase during transcription. The ribonucleotide to be added at the 3′ end of a growing RNA strand is specified by base pairing between the next base in the template DNA strand and the complementary incoming ribonucleoside triphosphate (rNTP). A phosphodiester bond is formed when RNA polymerase catalyzes a reaction between the 3′ oxygen of the growing strand and the α phosphate of a correctly base-paired rNTP. RNA strands are always synthesized in the 5′→3′ direction and are opposite in polarity to their template DNA strands. (b) Conventions for describing RNA transcription. Top: The DNA nucleotide where RNA polymerase begins transcription is designated +1. The direction the polymerase travels on the DNA is “downstream,” and downstream bases are marked with positive numbers. The opposite direction is “upstream,” and upstream bases are marked with negative numbers. Some important gene features lie upstream of the transcription start site, including the promoter sequence that localizes RNA polymerase to the gene. Bottom: The DNA strand that is being transcribed is the template strand; its complement is the nontemplate strand. The RNA being synthesized is complementary to the template strand and is therefore identical with the nontemplate strand sequence, except with uracil in place of thymine.

The energetics of the polymerization reaction strongly favor the addition of ribonucleotides to the growing RNA chain because the high-energy bond between the α and β phosphates of rNTP monomers is replaced by the lower-energy phosphodiester bond between nucleotides. The equilibrium for the reaction is driven further toward chain elongation by pyrophosphatase, an enzyme that catalyzes cleavage of the released PPi into two molecules of inorganic phosphate. Like the two strands in DNA, the template DNA strand and the growing RNA strand that is base-paired to it have opposite 5′→3′ directionality.

By convention, the site on the DNA template at which RNA polymerase begins transcription is numbered +1 (Figure 5-10b). Downstream denotes the direction in which a template DNA strand is transcribed; upstream denotes the opposite direction. Nucleotide positions in the DNA sequence downstream from a start site are indicated by a positive (+) sign; those upstream, by a negative (−) sign. Because RNA is synthesized 5′→3′, RNA polymerase moves down the template DNA strand in a 3′→5′ direction. The newly synthesized RNA is complementary to the template DNA strand; therefore, it is identical to the nontemplate DNA strand, with uracil in place of thymine.

Stages in Transcription To carry out transcription, RNA polymerase performs several distinct functions, as depicted in Figure 5-11. During transcription initiation, RNA polymerase, with the help of initiation factors (discussed later), recognizes and binds to a specific sequence of double-stranded DNA called a promoter (step 1). After binding, RNA polymerase and the initiation factors separate the DNA strands to make the bases in the template strand available for base pairing with the bases of the rNTPs that it will polymerize (step 2). Approximately 12–14 base pairs of DNA around the transcription start site on the template strand are separated, which allows the template strand to enter the active site of the enzyme. The active site is where catalysis of phosphodiester bond formation between rNTPs that are complementary to the template strand takes place. The 12–14-base-pair region of melted DNA in the polymerase is known as the transcription bubble. Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond (step 3).

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FIGURE 5-11 Three stages in transcription. During initiation of transcription, RNA polymerase forms a transcription bubble and begins polymerization of ribonucleotides (rNTPs) at the start site, which is located within the promoter region. Once a DNA region has been transcribed, the separated strands reassociate into a double helix. The nascent RNA is displaced from its template strand except at its 3′ end. The 5′ end of the RNA strand exits the RNA polymerase through a channel in the enzyme. Termination occurs when the polymerase encounters a specific termination sequence (stop site). See the text for details. For simplicity, the diagram depicts transcription of four turns of the DNA helix encoding some 40 nucleotides of RNA. Most RNAs are considerably longer, requiring transcription of a longer region of DNA.

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After several ribonucleotides have been polymerized, RNA polymerase dissociates from the promoter DNA and initiation factors (called σ-factors in bacteria, and general transcription factors in archaea and eukaryotes). During the strand elongation stage, RNA polymerase moves along the template DNA, opening the double-stranded DNA in front of its direction of movement and guiding the strands back together so that they reassociate at the upstream end of the transcription bubble (step 4). One ribonucleotide at a time is added by the polymerase to the 3′ end of the growing (nascent) RNA chain. During strand elongation, the enzyme maintains a melted region of approximately 14–20 base pairs in the transcription bubble. Approximately eight nucleotides at the 3′ end of the growing RNA strand remain base-paired to the template DNA strand in the transcription bubble. The elongation complex, comprising RNA polymerase, template DNA, and the nascent RNA strand, is extraordinarily stable. For example, RNA polymerase transcribes the longest known mammalian gene, containing about 2 million base pairs, without dissociating from the DNA template or releasing the nascent RNA. RNA synthesis occurs at a rate of about 1000–2000 nucleotides per minute at 37 °C, so the elongation complex must remain intact for more than 24 hours to ensure continuous synthesis of pre-mRNA from this very long gene.

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During transcription termination, the final stage in RNA synthesis, the completed RNA molecule is released from the RNA polymerase and the polymerase dissociates from the template DNA (step 5). Once it is released, an RNA polymerase is free to transcribe the same gene again or another gene.

Structure of RNA Polymerases The RNA polymerases of bacteria, archaea, and eukaryotic cells are fundamentally similar in structure and function. Bacterial RNA polymerases are composed of two related large subunits (β′ and β), two copies of a smaller subunit (α), and one copy of a fifth subunit (ω) that is not essential for transcription or cell viability, but that stabilizes the enzyme and assists in the assembly of its subunits. Archaeal and eukaryotic RNA polymerases have several additional small subunits associated with this core complex, which we describe in Chapter 9. Schematic diagrams of the transcription process generally show RNA polymerase bound to an unbent DNA molecule, as in Figure 5-11. However, x-ray crystallography and other studies of an elongating bacterial RNA polymerase indicate that the DNA bends at the transcription bubble (Figure 5-12).

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FIGURE 5-12 Bacterial RNA polymerase. This structure corresponds to the polymerase molecule in the elongation stage (step 4) of Figure 5-11. In this diagram, transcription is proceeding in the rightward direction. Arrows indicate where downstream DNA enters the polymerase and upstream DNA exits at an angle from the downstream DNA. The template strand is light violet, the nontemplate strand, dark violet; the nascent RNA, red. The RNA polymerase β′ subunit is gold; the β subunit, light yellow; and the α subunits visible from this angle, brown. Nucleotides complementary to the template DNA are added to the 3′ end of the nascent RNA strand on the right side of the transcription bubble. The newly synthesized nascent RNA exits the polymerase at the upstream side through a channel formed by the β subunit. The ω subunit is also visible from this angle.
[Data courtesy of Seth Darst; see N. Korzheva et al., 2000, Science 289:619–625, and N. Opalka et al., 2003, Cell 114:335–345.]