Arrangement of messenger RNA on Escherichia coli ribosomes with respect to 10 16S rRNA cross-linking sites.

Abstract

The arrangement of the mRNA on the Escherichia coli ribosome with respect to ribosomal RNA sites has been investigated by photochemical cross-linking experiments. mRNA analogues 51-54 nucleotides in length contained a Shine-Dalgarno sequence, a single codon for tRNA(Gly), and 4-thiouridine (s4U) in the 5' third of the mRNA (-20 to -12), in the middle third of the mRNA (-3 to +6), or in the 3' third of the mRNA (+20 to +26), where the position numbers are counted from the first nucleotide of the codon. Complexes were formed with these mRNAs and 70S ribosomes in the absence or presence of tRNA(Gly) and were irradiated. The extent of cross-linking and the identity of cross-linked rRNA sites were determined on agarose gels and by primer extension. 16S rRNA nucleotides A412, A532, G693 (weakly), U723, and U1381 (weakly) cross-linked with s4U in the 3' third; A532, G693, U723, A1167 (weakly), U1381, G818 (weakly), and A845 cross-linked with s4U in the middle; A532, G693, U723, A1167, G818 (weakly), and A845 cross-linked with s4U in the 5' third. All of these cross-links occur with tRNA independence. Cross-links at C1395 and A1196 occur for all three mRNAs with tRNA dependence. The pattern of these sites provides information about the order of the rRNA sites along the mRNA track, and they also point out the apparent overlapping neighborhoods for the mRNA track. Models for the track of the mRNA on the 30S subunit are considered to explain this pattern of interactions.