Tobin, C, Mandava, CS, Ehrenberg, M, Andersson, DI and Sanyal, S (2010) Ribosomes lacking protein S20 are defective in mRNA binding and subunit association. J. Mol. Biol. 397:767-76
The functional significance of ribosomal proteins is still relatively unclear. Here, we examined the role of small subunit protein S20 in translation using both in vivo and in vitro techniques. By means of lambda red recombineering, the rpsT gene, encoding S20, was removed from the chromosome of Salmonella enterica var. Typhimurium LT2 to produce a DeltaS20 strain that grew markedly slower than the wild type while maintaining a wild-type rate of peptide elongation. Removal of S20 conferred a significant reduction in growth rate that was eliminated upon expression of the rpsT gene on a high-copy-number plasmid. The in vitro phenotype of mutant ribosomes was investigated using a translation system composed of highly active, purified components from Escherichia coli. Deletion of S20 conferred two types of initiation defects to the 30S subunit: (i) a significant reduction in the rate of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in association with the 50S subunit. Both of these impairments were partially relieved by an extended incubation time with mRNA, fMet-tRNA(fMet), and initiation factors, indicating that absence of S20 disturbs the structural integrity of 30S subunits. Considering the topographical location of S20 in complete 30S subunits, the molecular mechanism by which it affects mRNA binding and subunit docking is not entirely obvious. We speculate that its interaction with helix 44 of the 16S ribosomal RNA is crucial for optimal ribosome function.
Colony Count, Microbial; Peptide Chain Initiation, Translational; RNA, Messenger/genetics; RNA, Messenger/metabolism; RNA, Transfer, Met/genetics; RNA, Transfer, Met/metabolism; Ribosomal Proteins/deficiency; Ribosome Subunits, Small/metabolism; Salmonella typhimurium/genetics; Salmonella typhimurium/growth & development; Salmonella typhimurium/metabolism
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