Sites of contact of mRNA with 16S rRNA and 23S rRNA in the Escherichia coli ribosome

Abstract

The locations of close encounter between ribosomal RNA (rRNA) and messenger RNA (mRNA) were determined by photochemical cross-linking experiments that employ an artificial mRNA, 51 nucleotides long, containing 14 U residues that were randomly substituted by 1-4 4-thiouridine (s4U) residues. The mRNA was bound to 70S ribosomes or 30S subunits and then was irradiated at 366 nm to activate cross-linking between the s4U residues and rRNA. Cross-linking occurred to both 16S rRNA and 23S RNA. The rRNA was then analyzed by a series of reverse transcriptase experiments to determine the locations of cross-linking. Twelve sites in the 16S rRNA and two sites in the 23S rRNA have been detected. In the 16S rRNA, two of the sites (U1381, C1395) are in the middle part of the secondary structure close to position C1400, and the remaining sites (G413, U421, G424; A532; G693; U723; A845; G1131/C1132; G1300; G1338) are distributed between six regions that are peripheral in the secondary structure. In the 23S rRNA, one site (U1065) is located in the GTPase center close to A1067, the site of thiostrepton-resistance methylation in domain II, and the other site (U887) is located a short distance away also in domain II. The distribution of these rRNA sites in the ribosome specifies an mRNA track that is consistent with other information. In addition, some of the contact points represent new constraints for the three-dimensional folding of the rRNA.