Molecular mechanism of tRNA binding by the Escherichia coli N7 guanosine methyltransferase TrmB

Sarah K Schultz; Kieran Meadows; Ute Kothe

doi:10.1016/j.jbc.2023.104612

Molecular mechanism of tRNA binding by the Escherichia coli N7 guanosine methyltransferase TrmB

J Biol Chem. 2023 May;299(5):104612. doi: 10.1016/j.jbc.2023.104612. Epub 2023 Mar 16.

Authors

Sarah K Schultz¹, Kieran Meadows², Ute Kothe³

Affiliations

¹ Alberta RNA Research and Training Institute (ARRTI), Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada; Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada.
² Alberta RNA Research and Training Institute (ARRTI), Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada.
³ Alberta RNA Research and Training Institute (ARRTI), Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada; Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada. Electronic address: ute.kothe@umanitoba.ca.

Abstract

Among the large and diverse collection of tRNA modifications, 7-methylguanosine (m⁷G) is frequently found in the tRNA variable loop at position 46. This modification is introduced by the TrmB enzyme, which is conserved in bacteria and eukaryotes. However, the molecular determinants and the mechanism for tRNA recognition by TrmB are not well understood. Complementing the report of various phenotypes for different organisms lacking TrmB homologs, we report here hydrogen peroxide sensitivity for the Escherichia coli ΔtrmB knockout strain. To gain insight into the molecular mechanism of tRNA binding by E. coli TrmB in real time, we developed a new assay based on introducing a 4-thiouridine modification at position 8 of in vitro transcribed tRNA^Phe enabling us to fluorescently label this unmodified tRNA. Using rapid kinetic stopped-flow measurements with this fluorescent tRNA, we examined the interaction of WT and single substitution variants of TrmB with tRNA. Our results reveal the role of S-adenosylmethionine for rapid and stable tRNA binding, the rate-limiting nature of m⁷G46 catalysis for tRNA release, and the importance of residues R26, T127, and R155 across the entire surface of TrmB for tRNA binding.

Keywords: 7-methylguanosine; RNA binding protein; RNA methylation; RNA modification; enzyme kinetics; fluorescence; presteady-state kinetics; transfer RNA (tRNA).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Escherichia coli* / metabolism
Guanosine
RNA, Transfer / metabolism
tRNA Methyltransferases* / chemistry

Substances

Guanosine
RNA, Transfer
tRNA Methyltransferases