The mRNA binding track in the Escherichia coli ribosome for mRNAs of different sequences

Abstract

Interactions between mRNA and rRNA on the 30S ribosomal subunit or 70S ribosome have been determined by photochemical cross-linking experiments using synthetic mRNA analogs substituted with 4-thiouridine. A set of RNA molecules containing different sequences has been used to determine the extent to which binding contacts are sequence dependent. The 16S rRNA and 23S rRNA nucleotides that form a part of the binding site have been identified by reverse transcription. The nucleotides are U1381, G1338, G1300, G1156, A845, U723, G693, A532, G497, U420, G413/A412, and G436 of 16S rRNA and U887 of 23S rRNA. Several additional nucleotides (U1065 of 23S rRNA and A1227, G818, G524, and G423 of 16S rRNA) are seen for some, but not all, of the mRNAs. Results obtained with two mRNAs containing the Shine-Dalgarno sequence were similar to those obtained with mRNAs lacking the Shine-Dalgarno sequence. Eight of these cross-linking sites were also seen when a mixture of RNA was used in which there are 12 random nucleotides preceding and seven random nucleotides succeeding an AUG codon. These results indicate that to a large extent placement of the mRNA in the ribosome does not depend upon its primary sequence.