Schaub, RE and Hayes, CS (2011) Deletion of the RluD pseudouridine synthase promotes SsrA peptide tagging of ribosomal protein S7. Mol. Microbiol. 79:331-41
RluD catalyses formation of three pseudouridine residues within helix 69 of the 50S ribosome subunit. Helix 69 makes important contacts with the decoding centre on the 30S subunit and deletion of rluD was recently shown to interfere with translation termination in Escherichia coli. Here, we show that deletion of rluD increases tmRNA activity on ribosomes undergoing release factor 2 (RF2)-mediated termination at UGA stop codons. Strikingly, tmRNA-mediated SsrA peptide tagging of two proteins, ribosomal protein S7 and LacI, was dramatically increased in ΔrluD cells. S7 tagging was due to a unique C-terminal peptide extension found in E. coli K-12 strains. Introduction of the rpsG gene (encoding S7) from an E. coli B strain abrogated S7 tagging in the ΔrluD background, and partially complemented the mutant's slow-growth phenotype. Additionally, exchange of the K-12 prfB gene (encoding RF2) with the B strain allele greatly reduced tagging in ΔrluD cells. In contrast to E. coli K-12 cells, deletion of rluD in an E. coli B strain resulted in no growth phenotype. These findings indicate that the originally observed rluD phenotypes result from synthetic interactions with rpsG and prfB alleles found within E. coli K-12 strains.
Codon, Terminator; Escherichia coli K12/genetics; Escherichia coli K12/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Gene Deletion; Genetic Complementation Test; Hydro-Lyases/genetics; Hydro-Lyases/metabolism; Intramolecular Transferases/genetics; Intramolecular Transferases/metabolism; Peptide Chain Termination, Translational; Peptide Termination Factors/metabolism; RNA, Bacterial/metabolism; RNA-Binding Proteins/metabolism; Ribosomal Proteins/metabolism
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