Ratcheting of a Viral Transcription Protein Machine along DNA without Backtracking
Update: 2018-02-24 08:44:40      Author: yangjuan@csrc.ac.cn

The RNA polymerases (RNAPs) play an essential role in gene expression as they transcribe information from DNA to RNA while synthesizing the RNA based on DNA. The viral RNAP from bacteriophage T7 is a prototypical single-subunit polymerase that is widely used in lab gene expression system as an efficient tool, yet rarely people understood underlying mechanisms well. T7 RNAP is indeed a smallest transcription machine, yet it is capable of working for all stages of transcription from initiation to elongation and to termination, without supports from any protein factor. It is in contrast with RNAPs from higher organisms, which constantly need assistances or regulations from a variety of protein factors. That being said, T7 RNAP sets a nice model system for physical investigations on transcription in a nutshell. In particular, the translocation mechanism of T7 RNAP on the transcribing DNA was controversial, and two basic working scenarios had been proposed, either as the thermally activated Brownian ratchet [1] or as the tightly coupled power-stroke engine [2].

11111111111111.png222222222222222222222.png Fig 1 The structural views of the active site of T7 RNAP elongation complexes before (pre) and after (post) the translocation (left), the methodological chart (middle), and the translocation schematics obtained (right).


To clarify the translocation mechanisms of T7 RNAP, Jin Yu’s group in CSRC recently performed extensive all-atom molecular dynamics simulations accumulated to ten microseconds and constructed the Markov state model (MSM) to reveal substantial structural dynamics of the transcription machine on DNA [3] (see Fig 1). Notably, they demonstrated that T7 RNAP moves along DNA via Brownian alike paths, facilitated by essential structural elements such as O-helix and Y-helix from the fingers subdomain. Interestingly, they found that the synthesizing RNA strand and its pairing template DNA move in non-synchronized manner. They further discovered that the O-helix could rotate to open even prior to translocation, not only to facilitate the translocation, but also to resist backtracking. The finding thus explains a long-standing puzzle on T7 RNAP not being detected with backtracking. Remarkably, they designed mutant T7 RNAPs via the O-helix to mimic structurally similar mitochondrial RNAPs for potential backtracking. Preliminary experimental tests from their experimental collaborator lab in PKU show survival of those mutants with more or less inhibited transcription activities, indicating the potential for backtracking. In summary, their work provides unprecedented detail and mechanistic insight into the translocation of a prototypical viral RNAP on DNA. The mechanism can be general for transcription machines with compact core structures. The rational re-design of the viral RNAP to acquire backtracking function preserved in other RNAP species also turns out to be highly promising.

 

References:

[1] Thomen P, Lopez PJ, and Heslot F. Unravelling the mechanism of RNA-polymerase forward motion by using mechanical force. Phys. Rev. Lett. 94,128102 (2005)

[2] Yin YW and Steitz TA.The structural mechanism of translocation and helicase activity in T7 RNA polymerase. Cell 116, 393 (2004)

[3] Da LT+,E C+, Shuai Y, Wu S, Su XD, and Yu J. T7 RNA polymerase translocation is facilitated by a helix opening on the fingers domain that may also prevent backtracking. Nucl. Acids Res. 45, 7909 (2017)  IF ~10.2



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