Structural analysis of the interaction of the tRNA modifying enzymes Tgt and QueA with a substrate tRNA

Abstract

The enzymes tRNA guanine-transglycosylase (Tgt) and S-adenosylmethionine :tRNA ribosyltransferase-isomerase (QueA) participate in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q) in Escherichia coli. Here we show by HPLC analysis and gel retardation that both enzymes interact with an in vitro transcribed tRNA(ASP) from yeast, specifically modified with a Q precursor molecule. RNase I footprinting experiments showed strong protein tRNA contacts in the anticodon stem-loop and a minor interaction with the dihydrouridine loop. This suggests that all identity elements for the recognition of Q-specific tRNAs are clustered in the anticodon region and explains earlier results that both enzymes accept a RNA microhelix with the sequence of an anticodon stem-loop as substrate.