| Popis: |
In all domains of life it is the multisubunit DNA-dependent RNA polymerase (RNAP) that governs transcription. In a single active centre, RNAP catalyses phosphodiester bond formation and its reversal pyrophosphorolysis, as well as hydrolysis of phosphodiester bonds of the nascent RNA. A plethora of factors and small molecules bind to RNAP in the secondary channel and regulate its activities. The mechanisms for such regulations remain unknown. In this study we investigated the role of the secondary channel binding transcription factors Gre, DksA, Gfh1, Rnk, and YacL, the alarmone ppGpp and the inhibitor Tagetitoxin. The main discovery of our study is that multiple modules substitute for each other in the active centre, in a controlled way, to change the catalytic properties of RNAP. We show that the trigger loop (TL), a mobile element of the active centre, participates in catalysis and stabilises the transition state of the phosphodiester bond synthesis. Transcription factor Gre substitutes for the TL to impose a highly effective hydrolytic activity exclusively to resolve backtracked complexes. Tagetitoxin modulates the TL and consequently the translocation of RNAP. DksA and ppGpp are not involved in the previously proposed functions during elongation, but possibly participate in transcription fidelity. Finally, we show that non-complementary NTPs may bind in a separate site in the active centre. |
