Role of hybrid tRNA-binding states in ribosomal translocation.
During translation, tRNAs must move rapidly to their adjacent sites in the ribosome while maintaining precise pairing with mRNA. This movement (translocation) occurs in a stepwise manner with hybrid-state intermediates, but it is unclear how these hybrid states relate to kinetically defined events of translocation. Here we analyze mutations at position 2394 of 23S rRNA in a pre-steady-state kinetic analysis of translocation. These mutations target the 50S E site and are predicted to inhibit P/E state formation. Each mutation decreases growth rate, the maximal rate of translocation (k(trans)), and the apparent affinity of EF-G for the pretranslocation complex (i.e., increases K(1/2)). The magnitude of these defects follows the trend A > G > U. Because the C2394A mutation did not decrease the rate of single-turnover GTP hydrolysis, the >20-fold increase in K(1/2) conferred by C2394A can be attributed to neither the initial binding of EF-G nor the subsequent GTP hydrolysis step. We propose that C2394A inhibits a later step, P/E state formation, to confer its effects on translocation. Replacement of the peptidyl group with an aminoacyl group, which is predicted to inhibit A/P state formation, decreases k(trans) without increasing K(1/2). These data suggest that movements of tRNA into the P/E and A/P sites are separable events. This mutational study allows tRNA movements with respect to both subunits to be integrated into a kinetic model for translocation.
Walker
Sarah E
SE
Department of Microbiology and Ohio State Biochemistry Program, Ohio State University, Columbus, OH 43210, USA.
Shoji
Shinichiro
S
Pan
Dongli
D
Cooperman
Barry S
BS
Fredrick
Kurt
K
D000225:Adenine; D002384:Catalysis; D049109:Cell Proliferation; D003596:Cytosine; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry* / Q000378:metabolism*; D020653:Peptide Elongation Factor G / Q000378:metabolism; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D009692:Nucleic Acid Heteroduplexes; D020653:Peptide Elongation Factor G; D006160:Guanosine Triphosphate; D003596:Cytosine; D012343:RNA, Transfer; D000225:Adenine