CONTACTS BETWEEN 16S RIBOSOMAL-RNA AND MESSENGER-RNA, WITHIN THE SPACER REGION SEPARATING THE AUG INITIATOR CODON AND THE SHINE-DALGARNO SEQUENCE - A SITE-DIRECTED CROSS-LINKING STUDY

mRNA analogues containing several 4-thiouridine (thio-U) residues at s elected positions were prepared by T7-transcription. The spacer region between the Shine-Dalgarno sequence and the AUG codon consisted of fo ur or eight bases with a single thio-U at a variable position; alterna tively, cro-mRNA analogues were used carrying the thio-U substituted s pacer sequence UUGU. The mRNAs were bound to E. coli ribosomes, and-af ter irradiation-the sites of crosslinking to 16S RNA were analysed. Th ree cross-links to the 16S RNA from the spacer region were observed, n amely to positions 665, 1360, and a site close to nucleotide 1530. The cross-links were formed in different amounts in the presence or absen ce of tRNA(fMet), and were observed from thio-ll residues located at v arious positions within the spacer sequence, although in the presence of tRNA they were in general stronger from positions close to the Shin e - Dalgarno end of the spacer. The cross-linking behaviour in this up stream area of the mRNA is thus rather different in character from the previously published pattern in the downstream area. From considerati ons of structural conservation in small subunit RNA, we propose that b oth the upstream and downstream cross-links to 16S RNA reflect a unive rsal mRNA path through the ribosome.