Mechano-chemical investigation revealing a jump-from-cavity product release model in gene transcription of a prototypical RNA polymerase
Update: 2016-02-02 13:37:38      Author: yangjuan@csrc.ac.cn

RNA polymerases (RNAPs) direct gene transcription by synthesizing RNA strand from template DNA. These protein enzymes are usually recognized to work as Brownian ratchet moving along double stranded DNA, with their mechanical movements and chemical reactions loosely coupled. That says, these protein machines move forward and backward without much free energy bias, while their forward movements are favored or backward movements are prevented upon RNA nucleotide incorporation, i.e., the chemical rectification. It is highly interesting to understand the physical basis of these physiologically important machines as they work under non-equilibrium condition, supported by chemical free energy from the enzymatic polymerization cycles.

Nevertheless, high-resolution structural studies on bacteriophage T7 RNAP model system had suggested an alternative power stroke mechano-chemical coupling scenario, which requires that the enzymatic reaction product (PPi) release tightly couples with or directly drives the enzyme movements. To resolve the conflicting views in the functional cycle of this prototypical transcription machine, a group of researchers led by Dr Jin Yu (tenure-track assistant professor) at CSRC complex system research division recently conducted intensive atomistic molecular dynamics (MD) simulations to investigate the PPi release mechanism of T7 RNAP [1]. The PPi release can be detected experimentally but happens too fast to be resolved in kinetic or structure dynamical detail.

By running a large number of nanosecond MD simulations, spreading around a wide range of conformational space of ~130,000 atoms of the solvated T7 RNAP, these researchers then constructed the simplified Markov state model (MSM) of the T7 RNAP product release process. In this way, they manage to efficiently organize the high-dimensional data to the physically tractable model, and to extract kinetic information for time scales up to tens of microseconds, which cannot be easily accessible by conventional MD. They found that the PPi release undergoes a jump-from-cavity activation process, unlikely to energetically support the polymerase translocation as being required for the power stroke scenario. In comparison, previous computational studies on multi-subunit RNAPs from higher organisms demonstrated instead charge facilitated hopping mechanisms of the product release. To explore the essential slow motions during the concerned process, the CSRC researchers further performed a small number of all-atom microsecond long MD simulations on this transcription machine. The PPi release does not appear to be tightly coupled to the rotational opening of an O-helix that is tied to the polymerase translocation. Hence, the study again disfavors the power stroke scenario, but favors the Brownian ratchet mechanism.

Remarkably, these researchers discovered a key residue Lys472 that is able to closely assist PPi to jump out of the cavity in T7 RNAP. The mechanism can apply general to many polymerases sharing conserved structural features with T7 RNAP, including viral or human mitochondria RNAPs, as well as a group of DNA polymerases that direct gene replication. Surprisingly, the lysine or arginine residue closely assisting the PPi release appears to be a universal module not only to the relatively simple single-subunit polymerases, but also to the much more complex multi-subunit RNAPs, even though the overall structural features and the full product release mechanisms vary significantly in the multi-subunit RNAPs.

In summary, this work has resolved unprecedented detail for structural dynamics of an essential process of a prototypical transcription machine. These researchers are continuing with similar computational efforts to further explore the translocation mechanisms of T7 RNAP, that combined would provide physical basis for the mechanochemistry of this prototype transcription machine.

This research has been supported by NSFC #11275022

Ref.: Lin-Tai Da, Chao E, Baogen Duan, Chuanbiao Zhang, Xin Zhou, and Jin Yu*. “A Jump-from-Cavity Pyrophosphate Ion Release Assisted by a Key Lysine Residue in T7 RNA Polymerase Transcription Elongation”, PLoS Computational Biology, 11 (11) e1004624 (2015). doi:10.1371/journal.pcbi.1004624

 

1.png

Fig 1. The product PPi release in T7 RNA polymerase transcription elongation. Left: The protein/DNA/RNA is shown in white/green/brown (O-helix in cyan). PPi in spheres (oxygen/phosphorus for red/brown). The key amino acids are shown with blue/red for positive/negative charged. In particular, Lys472 (blue) side chain swivels from the inside to the outside to assist the PPi release. Right: The simplified Markov state model with three states, showing PPi released from the inside cavity (top) to the outside protein surface (bottom).  


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