Initiation and Elongation

Initiation - After RNA polymerase has bound to a promoter and formed an open-promoter complex, the enzyme is ready to initiate synthesis of an RNA chain. One nucleoside triphosphate binding site on RNA polymerase is used during elongation. It binds any of the four common ribonucleoside triphosphates (rNTPs). Another binding site is used for initiation. It binds ATP and GTP preferentially. Thus, most mRNAs have a purine at the 5' end.

1. Chain growth begins with binding of the template-specified rNTP at the initiation-specific site of RNA polymerase (Figure 26.6, step 4),

2. The next nucleotide binds at the elongation-specific site.

3. Nucleophilic attack by the 3' hydroxyl of the first nucleotide on the (inner) phosphorus of the second nucleotide generates the first phosphodiester bond and leaves an intact triphosphate moiety at the 5' position of the first nucleotide.

Most initiations are abortive, with release of oligonucleotides 2 to 9 residues long. It is not yet clear why this happens.

and elongation - During transcription of the first 10 nucleotides, the subunit dissociates from the transcription complex, and the remainder of the transcription process is catalyzed by the core polymerase (Figure 26.6, steps 5 and 6). Once has dissociated, the elongation complex becomes quite stable. Transcription, as studied in vitro, can no longer be inhibited by adding rifampicin after this point, and virtually all transcription events proceed to completion.

Unwinding and rewinding - During elongation (Figure 26.6, steps 5 and 6), the core enzyme moves along the duplex DNA template and simultaneously unwinds the DNA, exposing a single-strand template for base pairing with incoming nucleotides and with the nascent transcript (the most recently synthesized RNA). It also rewinds the template behind the 3' end of the growing RNA chain. Figure 26.9 shows interactions in the transcription elongation complex. A view of transcriptional elongation is represented in Figure 26.8. In the model shown in Figure 26.8, about 18 base pairs of DNA are unwound to form a moving "transcription bubble." As one base pair becomes unwound in advance of the 3' end of the nascent RNA strand, one base pair becomes rewound near the trailing end of the RNA polymerase molecule. About 8 base pairs of the 3' end of the nascent transcript are hybridized to the template DNA strand.

Irregular movement - RNA polymerase often advances through DNA discontinuously, holding its position for several cycles of nucleotide addition and then jumping forward by several base pairs along the template. RNA polymerase "pauses" when it reaches DNA sequences that are difficult to transcribe in vitro, often sitting at the same site for several minutes before transcription is resumed. At such sites, RNA polymerase often translocates backward, and in the process the 3' end of the nascent transcript is displaced from the catalytic site of the enzyme. When this happens, a 3' "tail," is created which may be several nucleotides long and is not base-paired to the template, protruding downstream of the enzyme (Figure 26.10). In order for transcription to resume, the 3' end of the RNA must be positioned in the active site of the RNA polymerase. This is evidently the main function of the RNA 3' cleavage reactions catalyzed by the GreA and GreB proteins, which have been shown to stimulate a transcript cleavage activity intrinsic to the polymerase. These observations suggest that RNA polymerase movement generally moves forward until one of these special sequences is reached, or perhaps, until a transcription insertion error generates a DNA-RNA mispairing that weakens the hybrid and allows backtracking.

1. Regulation of Transcription by RNA Polymerase II

2. RNA Polymerase and GreA 3D Structures

3. Fundamental Mechanisms in the Initiation of Transcription