A summary of "Two distinct pathways of RNA polymerase backtracking determine the requirement for the Trigger Loop during RNA hydrolysis"

Transcription by RNAP is one of the essential steps in gene expression and it's regulated by several factors at all stages. The backtracking of RNAP occurs when the 3’ end of the transcript disengages from the template DNA and active center and the RNAP shifts backward along the template. Misincorporation events force the RNAP into a 1 base pair backtracked state.

                                                                                                 Evgeny Nudler, June 2012

Backtracking also occurs by several base pairs when the sequences of the nucleic acid scaffold thermodynamically favor backtracking as in the case of weak RNA:DNA hybrid, and certain sequences recognized by core RNAP that slows down translocation.

A backtracked conformation of the RNAP is highly stable and if not resolved can lead to genome instabilities, and hinder with gene expression.

In case of a sequence-dependent backtracked RNAP, the situation can be resolved by physically pushing the RNAP by coupling with translation. But misincorporated bases can be resolved only by RNA hydrolysis by RNAP active site.

The RNA hydrolysis in certain bacteria relies on Trigger Loop (TL), the catalytic domain of the active center. The TL folding stabilizes the transition state of the reaction through the amino acids H936 and R933. H936 plays a role in RNA hydrolysis as well as extension from mutational studies.

The study showed contrasting results. In case of 1 or 2 misincorporated bases when H936A substitution was done, it slowed down the reaction several fold but it.

Using Salinamide A which binds RNAP and inhibits its function by inhibiting TL function, this paper demonstrates the existence of 2 types of backtracked complexes in E.coli, one efficient in RNA cleavage and another one not and that TL is required to convert the backtracked complex into a cleavage efficient conformation.

From crystal structure, it is assumed that SAL inhibits RNAP by preventing the folding of TL. This is tested by designing a Transcription elongation complex with RNAP where TL is completely removed and using a control (WT) and 13 nucleotide-long RNA.

The binding of Salinamide is not template dependent and it inhibits Wildtype RNAP by nearly 340-fold whereas it is lower in ΔTL RNAP, about 70-fold.  Pyrophosphorolysis is strongly inhibited by SAL in wildtype as well but not as much in ΔTL RNAP. From this result, it is concluded that SAL inhibits Pyrophosphorolysis and this function is associated with TL. From structural prediction, it is concluded that SAL prevents the folding of TL into its active conformation.

It was observed that SAL strongly inhibited RNA hydrolysis in misincorporated EC suggesting the involvement of TL  in RNA cleavage as the rate of reaction is the same in misEC and ΔTL RNAP. This showed that TL had a role in RNA hydrolysis

Through experiments with sequence-dependent backtracked EC, RNAP which lacked TL (ΔTL RNAP), backtracked complex due to misincorporation (misEC) it was observed that TL plays a more significant role in RNA hydrolysis in case of misEC than in sequence-dependent EC, or ΔTL RNAP.

From Discussion: RNAP backtracking is different for misincorporated bases and when there is backtracking due to certain sequences. The contribution of the TL to correct 1bp backtracked complex is much less than that of a misincorporated base. In case of misincorporated bases the role of TL is critical as it helps orient the base into cleavage-active conformation.

Transcript-assisted hydrolysis of phosphodiester bonds appears to be conserved in bacteria as well as eukaryotes. But the role of TL seems to have diverged a lot. TL has a role of stabilizing the transition state during phosphodiester bond formation as well as in conformational changes to bring the misincorporated bases into a cleavage-active state but plays very little role in the actual catalysis, and in some rare cases plays a role in acid-base catalysis as well. This could be explained by the presence of Gre factors which take up the role of TL and speed up the process.