MAPPING THE CENTRAL FOLD OF TRANSFER RNA(2)(FMET) IN THE P-SITE OF THE ESCHERICHIA-COLI RIBOSOME

4-Thiouridine (s4U), a photoreactive analog of uridine, was randomly i ncorporated into tRNA2fMet precursor molecules by transcription with T 7 RNA polymerase. The s4U-containing transcripts were trimmed at their 5'-ends with RNase P RNA to yield mature tRNA2fMet. The photoreactive tRNA2fMet derivatives were aminoacylated and bound to the P site of 7 0S ribosomes from Escherichia coli in the presence of a poly(A,G,U) te mplate. Irradiation of the complexes at 300 nm resulted in the covalen t cross-linking of tRNA2fMet to ribosomal proteins and rRNAs within bo th the 50S and 30S subunits. The labeled proteins were identified as L 1, L27, and S19. 50S-subunit proteins L1 and L27 were attached to nucl eotide U17 or U17.1 within the D loop of tRNA2fMet, whereas 30S-subuni t protein S19 was cross-linked to nucleotide U47 in the variable loop. Both of these sites occur in or near the central fold of the tRNA. Th ese results permit us to map the D loop of P site-bound tRNA to the re gion between the central protuberance and the L1 ridge on the 50S ribo somal subunit, while the variable loop can be placed above the cleft o n the head of the 30S subunit.