Q FIGURE 4-10 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, displacing the nascent RNA 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.

polymerases require various protein factors, called general transcription factors, to help them locate promoters and initiate transcription. After binding to a promoter, RNA poly-merase melts the DNA strands in order to make the bases in the template strand available for base pairing with the bases of the ribonucleoside triphosphates that it will polymerize together. Cellular RNA polymerases melt approximately 14 base pairs of DNA around the transcription start site, which is located on the template strand within the promoter region (step 2). Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond (step 3 ).

After several ribonucleotides have been polymerized, RNA polymerase dissociates from the promoter DNA and general transcription factors. During the stage of strand elongation, RNA polymerase moves along the template DNA one base at a time, opening the double-stranded DNA in front of its direction of movement and hybridizing the strands behind it (Figure 4-10, step 4). One ribonucleotide at a time is added to the 3' end of the growing (nascent) RNA chain during strand elongation by the polymerase. The enzyme maintains a melted region of approximately 14 base pairs, called 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 growing (nascent) RNA strand, is extraordinarily stable. For example, RNA polymerase transcribes the longest known mammalian genes, containing «2 X 106 base pairs, without dissociating from the DNA template or releasing the nascent RNA. Since RNA synthesis occurs at a rate of about 1000 nucleotides per minute at 37 °C, the elongation complex must remain intact for more than 24 hours to assure continuous RNA synthesis.

During transcription termination, the final stage in RNA synthesis, the completed RNA molecule, or primary transcript, is released from the RNA polymerase and the polymerase dissociates from the template DNA (Figure 4-10, step 5). Specific sequences in the template DNA signal the bound RNA polymerase to terminate transcription. Once 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 (P' and p), two copies of a smaller subunit (a), and one copy of a fifth subunit (w) that is not essential for transcription or cell viability but 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 11. Schematic dia-

▲ FIGURE 4-11 Current model of bacterial RNA polymerase bound to a promoter. This structure corresponds to the polymerase molecule as schematically shown in step H of Figure 4-10. The p' subunit is in orange; p is in green. Part of one of the two a subunits can be seen in light blue; the m subunit is in gray. The DNA template and nontemplate strands are shown, respectively, as gray and pink ribbons. A Mg2+ ion at the active center is shown as a gray sphere. Numbers indicate positions in the DNA sequence relative to the transcription start site, with positive (+) numbers in the direction of transcription and negative (—) numbers in the opposite direction. [Courtesy of R. H. Ebright, Waksman Institute.]

grams of the transcription process generally show RNA poly-merase bound to an unbent DNA molecule, as in Figure 4-10. However, according to a current model of the interaction between bacterial RNA polymerase and promoter DNA, the DNA bends sharply following its entry into the enzyme (Figure 4-11).

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