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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 107; no. 24; pp. 10878 - 10883
• Main Authors: Yuzenkova, Yulia, Zenkin, Nikolay
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 15.06.2010; National Acad Sciences
• Subjects: Amino Acid Substitution; Biochemistry; Biological Sciences; Catalysis; Catalytic Domain; Chemical bases; Chemical bonding; Crystal structure; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; E coli; Enzyme Stability; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Gels; Hydrolysis; Kinetics; Macromolecular Substances; Magnesium; Models, Molecular; Mutagenesis, Site-Directed; Nucleotides; Proofreading; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Ribonucleic acid; RNA; RNA polymerase; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; Thermus; Thermus - enzymology; Thermus - genetics; Thermus aquaticus
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0914424107

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.