ROLE OF THE 3 CONSECUTIVE G-C BASE-PAIRS CONSERVED IN THE ANTICODON STEM OF INITIATOR TRANSFER-RNAS IN INITIATION OF PROTEIN-SYNTHESIS IN ESCHERICHIA-COLI

The three consecutive G:C base pairs, G29:C41, G30:C40, and G31:C39, a re conserved in the anticodon stem of virtually all initiator tRNAs fr om eubacteria, eukaryotes, and archaebacteria. We show that these G:C base pairs are important for function of the tRNA in initiation of pro tein synthesis in vivo, We changed these base pairs individually and i n combinations and analyzed the activities of the mutant Escherichia c oli initiator tRNAs in initiation in vivo, For assessment of activity of the mutant tRNAs in vivo, mutations in the G:C base pairs were coup led to mutation in the anticodon sequence from CAU to CUA, Mutations i n each of the G:C base pairs reduced activity of the mutant tRNA in in itiation, with mutation in the second G:C base pair having the most se vere effect. The greatly reduced activity of this C30:G40 mutant tRNA is not due to defects in aminoacylation or formylation of the tRNA or defects in base modification of the A37, next to the anticodon, which we had previously shown to be important for activity of the mutant tRN As in initiation, The anticodon stem mutants are most likely affected specifically at the step of binding to the ribosomal P site, The patte rn of cleavages in the anticodon loop of mutant tRNAs by S1 nuclease i ndicate that the G:C base pairs may be involved directly in interactio ns of the tRNA with components of the P site on the ribosome rather th an indirectly by inducing a particular conformation of the anticodon l oop critical for function of the tRNA in initiation.