Variations in the inner ribosomal landscape determining the topology of nascent protein transport have been studied by three-dimensional cryo-electron microscopy of erythromycin-resistant Escherichia coli 70S ribosomes. Significant differences in the mouth of the 50S subunit tunnel system visualized in the present study support a simple steric-hindrance explanation for the action of the drug. Examination of ribosomes in different functional states suggests that opening and closing of the main tunnel are dynamic features of the large subunit, possibly accompanied by changes in the L7/L12 stalk region. The existence and dynamic behavior of side tunnels suggest that ribosomal proteins L4 and L22 might be involved in the regulation of a multiple exit system facilitating cotranslational processing (or folding or directing) of nascent proteins.
Direct evidence is presented for a conformational switch in 16S ribosomal RNA (rRNA) that affects tRNA binding to the ribosome and decoding of messenger RNA (mRNA). These data support the hypothesis that dynamic changes in rRNA structure occur during translation. The switch involves two alternating base-paired arrangements apparently facilitated by ribosomal proteins S5 and S12, and produces significant changes in the rRNA structure. Chemical probing shows reciprocal enhancements or protections at sites in 16S rRNA that are at or very near sites that were previously crosslinked to mRNA. These data indicate that the switch affects codon-anticodon arrangement and proper selection of tRNA at the ribosomal A site, and that the switch is a fundamental mechanism in all ribosomes.
Watson‐Crick base pairing is shown to occur between the mRNA and nucleotides near the 3′ end of 16S rRNA during the elongation phase of protein synthesis in Escherichia coli. This base‐pairing is similar to the mRNA‐rRNA interaction formed during initiation of protein synthesis between the Shine and Dalgarno (S‐D) nucleotides of ribosome binding sites and their complements in the 1540‐1535 region of 16S rRNA. mRNA‐rRNA hybrid formation during elongation had been postulated to explain the dependence of an efficient ribosomal frameshift on S‐D nucleotides precisely spaced 5′ on the mRNA from the frameshift site. Here we show that disruption of the postulated base pairs by single nucleotide substitutions, either in the S‐D sequence required for shifting or in nucleotide 1538 of 16S rRNA, decrease the amount of shifting, and that this defect is corrected by restoring complementary base pairing. This result implies that the 3′ end of 16S rRNA scans the mRNA very close to the decoding sites during elongation.
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