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Oligonucleotides such as G-A-G-G, which are complementary to the C-U-C-C region at the 3' end of 16-S RNA, inhibit the R17-RNA-dependent binding of the initiator tRNA (fMet-rRNA) to 30-S ribosomal subunits. However, if phage RNA is replaced by A-U-G, the same oligonucleotides stimulate the binding of fMet-tRNA to the 30-S subunits. This indicates that the formation of the RNA x RNA hybrid acts as a positive control signal for the selection of the initiator tRNA by the 30-S-subunit x mRNA complex. Tetranucleotides of the type A-U-G-N (where N = A, G, C or U) stimulated the IF-2-dependent binding of fMet-tRNA to the 30-S subunit more effectively than A-U-G, with A-U-G-R better than A-U-G-Y (where R is a purine nucleoside and Y is a pyrimidine nucleoside). Since the 3'-terminal adenosine in A-U-G-A can be replaced by 6-deamino-adenosine, a stacking type of interaction between U-33 of tRNA and N of A-U-G-N should additionally stabilize the codon-anticodon complex. The situation is strictly reversed for 70-S ribosomes where A-U-G is the best codon followed by A-U-G-U, A-U-G-C, A-U-G-G and A-U-G-A. Replacement of GTP by guanosine 5'-[beta, gamma-methylene]triphosphate (GuoPP[CH2]P] results in A-U-G-A becoming more efficient than A-U-G as the codon for the binding of fMet-tRNA to 70-S ribosomes. This indicates that IF-2 and GTP hold the anticodon of the fMet-tRNA in a conformation capable of binding to a tetranucleotide codon. GTP hydrolysis and release of IF-2 from the 70-S ribosome results in a change of the tertiary structure of fMet-tRNA as a consequence of which the initiator tRNA reassumes the conformation which preferentially binds to A-U-G.

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 TWC 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 tRNAMet 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 Vmax value is decreased by onehalf. (ii) tRNAfmet 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-tRNAPet species with U54 and rT54, respectively, function equally well in an in vitro initiation system containing AUG, initiation factors, and 70s ribosomes. The unformylated MetAtRNAmet species, however, differ from each other: "Met-tRNAm't rT" is inactive, whereas the U54 counterupart effectively forms the initiation complex. (iii) Two isoacceptors,tRNAlhe and tRNA he, were obtained from B. subtilis. tRNAIhe accumulates only under special growth conditions and is an incompletely modified precursor oftRNA2he: in the first position of the anticodon, guanosine replaces Gm, and next to the 3' end of the anticodon (isopentenyl)adenosine replaces 2-thiomethyl-N6-(isopentenyl)adenosine. Both tRNA's behave identically in aminoacylation kinetics. In the factor-dependent AUGU3-directed fornation of fMet-Phe, the undermodified tRNAphe is always less efficient at Mg2+ concentrations between 5 and 15 mM than its mature counterpart.

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