Kinetic properties of Escherichia coli ribosomes with altered forms of S12.
E. coli ribosomes with alterations in S12 leading to streptomycin resistance (SmR), dependence (SmD) and pseudodependence (SmP) were studied with the quench-flow technique. Kinetic changes at the various steps of the elongation cycle were identified. The rate of hydrolysis of GTP in the ternary complex in the ribosomal A-site is decreased drastically in SmD and moderately in SmP in relation to wild-type ribosomes. Addition of streptomycin restores much of the wild-type behaviour. The SmD, SmP and SmR ribosomes have an enhanced GTP-hydrolysis idling reaction on EF-Tu, which is correlated with how aggressive proofreaders these ribosomes are in steady-state assays. We use our in vitro findings to discuss the in vivo physiology of these mutants as well as mechanistic features of E. coli translation.
Bilgin
N
N
University of Uppsala, Department of Molecular Biology, Sweden.
Claesens
F
F
Pahverk
H
H
Ehrenberg
M
M
D004926:Escherichia coli / Q000378:metabolism; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D010441:Peptide Chain Elongation, Translational; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010458:Peptidyl Transferases / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D013307:Streptomycin / Q000494:pharmacology
D012269:Ribosomal Proteins; C034212:ribosomal protein S12; D006160:Guanosine Triphosphate; D010458:Peptidyl Transferases; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu; D013307:Streptomycin