3 MODIFIED NUCLEOSIDES PRESENT IN THE ANTICODON STEM AND LOOP INFLUENCE THE IN-VIVO AA-TRANSFER-RNA SELECTION IN A TRANSFER-RNA-DEPENDENT MANNER

In Salmomella typhimurium seven tRNA species specific for leucine, pro line and arginine have 1-methylguanosine (m(1)G) next to and 3' of the anticodon (position 37 of tRNA), five tRNA species specific for pheny lalanine, serine, tyrosine, cysteine and tryptophan have 2-methylthio- N-6-(cis-hydroxy)isopentenyladenosine (ms(2)io(6)A) in the same positi on of the tRNA, and four tRNA species, specific for leucine and prolin e, have pseudouridine (Psi) as the last 3' nucleotide in the anticodon loop (position 38) or in the anticodon stem (positions 39 and 40). Mu tants deficient in the synthesis of these modified nucleosides have be en used to study their role in the first step of translation elongatio n, i.e. the aa-tRNA selection step in which the ternary complex (EF-Tu -GTP-aa-tRNA) binds at the cognate codon in the A-site on the mRNA pro grammed ribosome. We have found that the Psi present in the anticodon loop (position 38) stimulates the selection of tRNA specific for leuci ne whereas Psi in the anticodon stem did not affect the selection of t RNA specific for proline. The m(1)G37 strongly stimulates the rate of selection of the three tRNA species specific for proline and one tRNA species specific for arginine but has only minor or no effect on the s election of the three tRNA species specific for leucine. Likewise, the ms(2)io(6)A, present in the same position as m(1)G37 but in another s ubset of tRNA species, stimulates the selection of tRNA specific for t yrosine, stimulates to some extent also tRNA species specific for cyst eine and tryptophan, but has no influence on the rate of selection of tRNA specific for phenylalanine. We conclude that function of m(1)G an d ms(2)io(6)A present next to and 3' of the anticodon influences the i n vivo aa-tRNA selection in a tRNA-dependent manner. (C) 1997 Academic Press Limited.