Molecular mechanism of tRNA binding by theEscherichia coliN7 guanosine methyltransferase TrmB

Abstract

Among the large and diverse collection of tRNA modifications, 7-methylguanosine 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. Complementing the report of various phenotypes for different organisms lacking TrmB homologs, we report here hydrogen peroxide sensitivity for theEscherichia coli ΔtrmBknockout strain. To gain insight into the molecular mechanism of tRNA binding byE. coliTrmB in real-time, we developed a new assay based on introducing a 4-thiouridine modification at position 8 ofin vitrotranscribed tRNAPheenabling us to fluorescently labelled this unmodified tRNA. Using rapid kinetic stopped-flow measurements with this fluorescent tRNA, we examined the interaction of wild-type and single substitution variants of TrmB with tRNA. Our results reveal the role of SAM 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.

Different modification pathways for m1A58 incorporation in yeast elongator and initiator tRNAs

“…In a circuit of modifications, the observed effect of the initial modification on the subsequent enzyme is reflected in an increased turnover rate, meaning either a better substrate binding, or a better catalytic efficiency, or a better product release, depending on the enzyme considered (56). For RNA modification enzymes, the rate-determining step of the reaction was reported to be the catalytic step (57,58), the product release (59,60), or conformational changes of both the RNA and protein, most probably to accommodate the target nucleotide into the active site (61).…”

“…In a circuit of modifications, the observed effect of the initial modification on the subsequent enzyme is reflected in an increased turnover rate, meaning either a better substrate binding, or a better catalytic efficiency, or a better product release, depending on the enzyme considered (56). For RNA modification enzymes, the rate-determining step of the reaction was reported to be the catalytic step (57,58), the product release (59,60), or conformational changes of both the RNA and protein, most probably to accommodate the target nucleotide into the active site (61). In a more general description, one could either consider that the initial modification directly acts as a recognition element for the subsequent modification enzyme, or that the initial modification alters the local or overall structure of the tRNA substrate, thereby presenting a structural conformation that is more efficiently modified by the subsequent enzyme.…”

Modifications in the T arm of tRNA globally determine tRNA maturation, function and cellular fitness

“…To compare the fraction of thiolated tRNAs to non-thiolated tRNAs, 5 µg total RNA was separated on a 20 µM [(N-acryloylamino)phenyl]mercuric chloride (APM, Toronto Research Chemicals) 7 M urea 12% polyacrylamide gel 28 . To reduce APM-thiol linkages, APM gels were soaked in 0.2 M β-mercaptoethanol for one hour prior to transfer.…”

“…In particular, we examined whether s 4 U8 (Fig. 3G) abundance is dependent on presence of TrmA or TruB by using urea-PAGE containing a phenylmercury compound that decreases the migration velocity of sulfur-containing tRNAs, wherein we detected specific tRNAs by Northern blotting 28 . For many tRNAs, a high level (near 100%) of tRNA thiolation is observed in the absence and presence of TrmA and TruB enzymes (Extended Data Fig.…”