Function of Modified Nucleosides 7-Methylguanosine, Ribothymidine, and 2-Thiomethyl- N 6 -(Isopentenyl)adenosine in Procaryotic Transfer Ribonucleic Acid

Abstract

To elucidate subtle functions of transfer ribonucleic acid (tRNA) modifications in protein synthesis, pairs of tRNA's that differ in modifications at specific positions were prepared from Bacillus subtilis . The tRNA's differ in modifications in the anticodon loop, the extra arm, and the T C loop. The functional properties of these species were compared in aminoacylation, as well as in initiation and peptide bond formation, at programmed ribosomes. These experiments demonstrated the following. (i) In tRNA f Met the methylation of guanosine 46 in the extra arm to 7-methylguanosine by the 7-methylguanosine–forming enzyme from Escherichia coli changes the aminoacylation kinetics for the B. subtilis methionyl-tRNA synthetase. In repeated experiments the V max value is decreased by one-half. (ii) tRNA f Met species with ribothymidine at position 54 (rT54) or uridine at position 54 (U54) were obtained from untreated or trimethoprim-treated B. subtilis . The formylated fMet-tRNA f Met species with U54 and rT54, respectively, function equally well in an in vitro initiation system containing AUG, initiation factors, and 70s ribosomes. The unformylated Met-tRNA t Met species, however, differ from each other: “Met-tRNA f Met rT” is inactive, whereas the U54 counter-upart effectively forms the initiation complex. (iii) Two isoacceptors, tRNA 1 Phe and tRNA 2 Phe , were obtained from B. subtilis . tRNA 1 Phe accumulates only under special growth conditions and is an incompletely modified precursor oftRNA 2 Phe : in the first position of the anticodon, guanosine replaces Gm, and next to the 3′ end of the anticodon (isopentenyl)adenosine replaces 2-thiomethyl- N 6 -(isopentenyl)adenosine. Both tRNA's behave identically in aminoacylation kinetics. In the factor-dependent AUGU 3 -directed formation of fMet-Phe, the undermodified tRNA 1 Phe is always less efficient at Mg 2+ concentrations between 5 and 15 mM than its mature counterpart.