Mutations at the accommodation gate of the ribosome impair RF2-dependent translation termination.
During protein synthesis, aminoacyl-tRNA (aa-tRNA) and release factors 1 and 2 (RF1 and RF2) have to bind at the catalytic center of the ribosome on the 50S subunit where they take part in peptide bond formation or peptidyl-tRNA hydrolysis, respectively. Computer simulations of aa-tRNA movement into the catalytic site (accommodation) suggested that three nucleotides of 23S rRNA, U2492, C2556, and C2573, form a "gate" at which aa-tRNA movement into the A site is retarded. Here we examined the role of nucleotides C2573 of 23S rRNA, a part of the putative accommodation gate, and of the neighboring A2572 for aa-tRNA binding followed by peptide bond formation and for the RF2-dependent peptide release. Mutations at the two positions did not affect aa-tRNA accommodation, peptide bond formation, or the fidelity of aa-tRNA selection, but impaired RF2-catalyzed peptide release. The data suggest that the ribosome is a robust machine that allows rapid aa-tRNA accommodation despite the defects at the accommodation gate. In comparison, peptide release by RF2 appears more sensitive to these mutations, due to slower accommodation of the factor or effects on RF2 positioning in the A site.
Burakovsky
Dmitry E
DE
Department of Chemistry, Moscow State University, Moscow, 119899, Russia.
Sergiev
Petr V
PV
Steblyanko
Maria A
MA
Kubarenko
Andriy V
AV
Konevega
Andrey L
AL
Bogdanov
Alexey A
AA
Rodnina
Marina V
MV
Dontsova
Olga A
OA
D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000378:metabolism*; D014176:Protein Biosynthesis; D012338:RNA, Ribosomal, 23S / Q000235:genetics* / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D012338:RNA, Ribosomal, 23S; C041460:peptide chain termination release factor 2; D012343:RNA, Transfer