Kinetics of stop codon recognition by release factor 1.
Recognition of stop codons by class I release factors is a fundamental step in the termination phase of protein synthesis. Since premature termination is costly to the cell, release factors have to efficiently discriminate between stop and sense codons. To understand the mechanism of discrimination between stop and sense codons, we developed a new, pre-steady state kinetic assay to monitor the interaction of RF1 with the ribosome. Our results show that RF1 associates with similar association rate constants with ribosomes programmed with stop or sense codons. However, dissociation of RF1 from sense codons is as much as 3 orders of magnitude faster than from stop codons. Interestingly, the affinity of RF1 for ribosomes programmed with different sense codons does not correlate with the defects in peptide release. Thus, discrimination against sense codons is achieved with both an increase in the dissociation rates and a decrease in the rate of peptide release. These results suggest that sense codons inhibit conformational changes necessary for RF1 to stably bind to the ribosome and catalyze peptide release.
Hetrick
Byron
B
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0314, USA.
Lee
Kristin
K
Joseph
Simpson
S
D003062:Codon / Q000032:analysis / Q000378:metabolism; D018388:Codon, Terminator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D007700:Kinetics; D008856:Microscopy, Fluorescence; D010443:Peptide Chain Termination, Translational / Q000235:genetics / Q000502:physiology*; D010454:Peptide Termination Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003062:Codon; D018388:Codon, Terminator; D010454:Peptide Termination Factors; D012333:RNA, Messenger