| Modified nucleotides in 16S ribosomal RNA are clustered at its functional center. Helix 31 (970 loop) of Escherichia coli 16S rRNA consists of 2-methylguanosine at position 966 (m2G966) and 5-methylcytidine at position 967 (m5C967). The modifications in h31 are phylogenetically conserved within each domain. Helix 31 is located at the ribosomal P site and interacts with tRNA, ribosomal proteins, and initiation factors. Single mutations at positions 966 or 967 produced hyperactive ribosomes that made more protein than wild-type ribosomes. Double mutations at both positions, however, decreased ribosome function below wild-type levels. Using methylase knock-out strains, we showed that hyperactivity in the single mutants is due to loss of nucleotide methylation. Ribosome activity in the hyperactive mutants returns to wild-type levels when initiation factor 3 is over-expressed in the mutants. To evaluate the role of modification in initiation, the start codon of the reporter genes was substituted with non-canonical start codons. Spurious initiation was not observed from any of the single or double mutants of m2G966 and m5C967, suggesting that hyperactivity is not due to a defect in the selection of non-canonical initiator tRNA during initiation. Green fluorescent protein translated by these hyperactive ribosomes was purified and analyzed to see if the loss of modification increased the error rate of the ribosomes. Loss of modification at G966 or C967 significantly increased the level of mistranslation by the mutant ribosomes. The methylation-deficient ribosomes also read through stop codons at a higher frequency than wild-type ribosomes. These data suggest that the loss of modifications in h31 reduce translational fidelity during protein synthesis. A series of phage display experiments were performed to identify peptides that specifically bind to h31 containing m2G966 and m5C967. The two peptides, CVRPFAL and TLWDLIP were generated as protein fusions. The fusion proteins showed modest affinity to h31, with dissociation constants in the high nanomolar range. These peptides are being further characterized and derivatives are being synthesized to determine their potential as domain-specific antimicrobial drug leads. |
