Central role of the RNA polymerase trigger loop in intrinsic RNA hydrolysis.
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 Mg(2+) 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 (beta' 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 beta' 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.
Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4AX, United Kingdom.
Yulia
Y
Yuzenkova
Nikolay
N
Zenkin
D019943:Amino Acid Substitution; D020134:Catalytic Domain; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013824:Thermus / Q000201:enzymology / Q000235:genetics
D046911:Macromolecular Substances; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases