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Nucleic Acids Res. 2014 Jan;42(1):544-52. doi: 10.1093/nar/gkt877. Epub 2013 Oct 1.

Interplay between the trigger loop and the F loop during RNA polymerase catalysis.

Author information

1
Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia, Molecular Biology Department, Biological Faculty, Moscow State University, Moscow 119991 Russia, Department of Biological DNA Modification, Institute of Biotechnology, Vilnius University, Vilnius 02241, Lithuania and Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA.

Abstract

The trigger loop (TL) in the RNA polymerase (RNAP) active center plays key roles in the reactions of nucleotide addition and RNA cleavage catalyzed by RNAP. The adjacent F loop (FL) was proposed to contribute to RNAP catalysis by modulating structural changes in the TL. Here, we investigate the interplay between these two elements during transcription by bacterial RNAP. Thermodynamic analysis of catalysis by RNAP variants with mutations in the TL and FL suggests that the TL is the key element required for temperature activation in RNAP catalysis, and that the FL promotes TL transitions during nucleotide addition. We reveal characteristic differences in the catalytic parameters between thermophilic Thermus aquaticus and mesophilic Deinococcus radiodurans RNAPs and identify the FL as an adaptable element responsible for the observed differеnces. Mutations in the FL also significantly affect the rate of intrinsic RNA cleavage in a TL-dependent manner. In contrast, much weaker effects of the FL and TL mutations on GreA-assisted RNA cleavage suggest that the FL-dependent TL transitions are not required for this reaction. Thus, functional interplay between the FL and TL is essential for various catalytic activities of RNAP and plays an adaptive role in catalysis by thermophilic and mesophilic enzymes.

PMID:
24089145
PMCID:
PMC3874190
DOI:
10.1093/nar/gkt877
[Indexed for MEDLINE]
Free PMC Article

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