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Central role of the RNA polymerase trigger loop in intrinsic RNA hydrolysis

Author:
Yuzenkova, Yulia, Zenkin, Nikolay
Source:
Proceedings of the National Academy of Sciences of the United States of America 2010 v.107 no.24 pp. 10878-10883
ISSN:
0027-8424
Subject:
DNA-directed RNA polymerase, Escherichia coli, RNA, Thermus aquaticus, bacteria, catalytic activity, histidine, hydrolysis, magnesium
Abstract:
The active center of RNA polymerase can hydrolyze phosphodiester bonds in nascent RNA, a reaction thought to be important for proofreading of transcription. The reaction proceeds via a general two Mg²⁺ mechanism and is assisted by the 3' end nucleotide of the transcript. Here, by using Thermus aquaticus RNA polymerase, we show that the reaction also requires the flexible domain of the active center, the trigger loop (TL). We show that the invariant histidine (β' His1242) of the TL is essential for hydrolysis/proofreading and participates in the reaction in two distinct ways: by positioning the 3' end nucleotide of the transcript that assists catalysis and/or by directly participating in the reaction as a general base. We also show that participation of the β' His1242 of the TL in phosphodiester bond hydrolysis does not depend on the extent of elongation complex backtracking. We obtained similar results with Escherichia coli RNA polymerase, indicating that the function of the TL in phosphodiester bond hydrolysis is conserved among bacteria.
Agid:
2369191