Structural requirements for the formation of 1-methylguanosine in vivo in tRNAGGGPro of Salmonella typhimurium

Abstract

Maturation of tRNA and rRNA and the assembly of the ribosome in all organisms occurs in vivo in a complex pathway in which various proteins such as endo- and exonucleases, tRNA and rRNA modifying enzymes and ribosomal proteins, act concomitantly and temporarily during the maturation process. One class of RNA binding proteins are the tRNA modifying enzymes, which catalyse the formation of various modified nucleosides present in tRNA. Here we analyse the consequences of various alterations in a tRNA on the formation of modified nucleosides in the tRNA and the aminoacylation of it under true in vivo conditions, i.e. in a cell with normal amounts of the tRNA substrate and the tRNA binding protein. We have devised a selection method to obtain mutants of tRNAGGGPro in Salmonella typhimurium that may no longer be a substrate in vivo for the RNA(m1G37)methyltransferase. These mutant tRNAs were purified from cells in balanced growth by a solid phase hybridisation technique and the presence of 1-methylguanosine (m1G) in position 37 next to the anticodon was monitored. Of 13 different mutant tRNAGGGPro species analysed, eight of them had a drastically reduced level of m1G. Some of these mutant tRNA species had alterations far from the nucleotide G37 modified by the enzyme; e.g. base-pair disruptions in the first, fourth and eighth (last) base-pair of the acceptor stem, in the D-stem, and in the top of the anticodon stem. The structure of all the mutant tRNAGGGPro species must deviate from the wild-type form, since they all induced +1 frameshifting. Still, tRNAGGGPro from five of the mutants had normal levels of m1G. Thus, only a subset of mutations, all inducing an altered tRNA structure, resulted in m1G deficiency. However, those alterations in tRNAGGGPro,which influenced the tRNA(m1G37)methyltransferase activity, did not affect in vivo the formation of four other modified nucleosides and the aminoacylation of tRNAGGGPro,demonstrating the extreme dependence of the tRNA(m1G37)methyltransferase on an almost perfect three-dimensional structure of the tRNA. We discuss that the conformation of the anticodon loop may be a major determining element for the formation of m1G37 in vivo.