STRUCTURAL REQUIREMENTS FOR THE FORMATION OF 1-METHYLGUANOSINE IN-VIVO IN TRNA(GGG)(PRO) OF SALMONELLA-TYPHIMURIUM

Maturation of tRNA and rRNA and the assembly of the ribosome in all or ganisms occurs in vivo in a complex pathway in which various proteins such as endo- and exonucleases, tRNA and rRNA modifying enzymes and ri bosomal proteins, act concomitantly and temporarily during the maturat ion 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 alteratio ns 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 protei n. We have devised a selection method to obtain mutants of tRNA(GGG)(P ro), in Salmonella typhimurium that may no longer be a substrate in vi vo for the tRNA(m(1)G37)methyltransferase. These mutant tRNAs were pur ified from cells in balanced growth by a solid phase hybridisation tec hnique and the presence of 1-methylguanosine (m(1)G) in position 37 ne xt to the anticodon was monitored. Of 13 different mutant tRNA(GGG)(Pr o) species analysed, eight of them had a drastically reduced level of m(1)G. Some of these mutant tRNA species had alterations far from the nucleotide G37 modified by the enzyme; e.g. base-pair disruptions in t he 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 tRNA(GGG)(Pro) species must deviate from the wild-type for m, since they all induced +1 frameshifting. Still, tRNA(GGG)(Pro) from five of the mutants had normal levels of m(1)G. Thus, only a subset o f mutations, all inducing an altered tRNA structure, resulted in m(1)G deficiency. However, those alterations in tRNA(GGG)(Pro), which influ enced the tRNA(m(1)G37)Pro methyltransferase activity, did not affect in vivo the formation of four other modified nucleosides and the amino acylation of tRNA(GGG)(Pro) demonstrating the extreme dependence of th e tRNA(m(1)G37)methyltransferase on an almost perfect three-dimensiona l structure of the tRNA. We discuss that the conformation of the antic odon loop may be a major determining element for the formation of m(1) G37 in vivo. (C) 1997 Academic Press Limited.