The Mechanism: Elongation
The pre-initiation complex is completely formed once all general transcription factors, RNAPII, and the multi-protein mediator complex bind to the promoter. This complex is necessary not only to prepare RNAPII for transcription, but may also serve as a precursor for the elongation complex.
The switch from initiation to elongation involves the following processes: promoter clearance, promoter escape, and elongation processivity (1). In order for these to occur, the pre-initiation complex must swap out its cofactors for new cofactors. The phosphorylation of the CTD of RNAPII is also required, because this domain recruits 3’ capping enzymes that cap newly created mRNA after it has left the enzyme.
Both promoter clearance and promoter escape are regarded as regulatory processes. This means they serve as checkpoints in which certain requirements must be met in order for transcription to continue. After the pre-initiation complex has been formed, a variety of positive and negative elongation factors compete with each other to bind the complex. Only when the positive factors win the competition, the checkpoint requirements can be met, pushing transcription from initiation to elongation.
Elongation factors
One of the negative elongation factors, transcription release factor 2 (Factor 2), prevents RNAPII from producing long mRNA transcripts and instead causes the release of short nascent transcripts. Two others, DSIF and NELF, also prevent elongation from occurring when they associate with a transcription inhibitor known as DRB.
Three positive elongation factors, P-TEFb, TFIIF, and TFIIH, compete against the negative elongation factors to promote elongation of transcription. P-TEFb promotes the phosphorylation of the CTD in elongation by specifically countering the effects of DSIF and NELF. In addition to recruiting RNAPII to the pre-initiation complex, TFIIF functions in both reducing RNAPII’s production of abortive transcripts and preventing RNAPII from pausing during elongation. Other than TFIIH’s phosphorylation activity, its helicase-associated activity also plays an important role in promoter clearance as described earlier.
In order to ensure the processivity of the elongation stage, the elongation complex binds TFIIS, Elongin, ELL, Cockayne Syndrome B protein, Tat, and many other elongation factors. TFIIS is an important factor as it is known to prevent RNAPII from pausing during elongation. When the enzyme is paused due to an obstruction or blockage in its path, TFIIS cleaves the nascent mRNA to free the enzyme and help it begin transcribing again. The other elongation factors mentioned perform similar functions to TFIIS or enhance elongation rates of RNAPII.
During elongation, RNAPII must move down the DNA one nucleotide at a time in order to expose a new site for synthesis. This process is called translocation. In another section of this review, the description of a-amanitin, an inhibitor of translocation in transcription, explains this process in detail (9). RNAPII can also proofread its work by backtracking or moving backwards to replace a misincorporated base pair. Another description in this review, explains how mRNA transcripts themselves stimulate their own correction (13).