Histidine 197 in release factor 1 is essential for a site binding and peptide release.
Class I peptide release factors 1 and 2 (RF1 and RF2, respectively) recognize the stop codons in the ribosomal decoding center and catalyze peptidyl-tRNA hydrolysis. High-fidelity stop codon recognition by these release factors is essential for accurate peptide synthesis and ribosome recycling. X-ray crystal structures of RF1 and RF2 bound to the ribosome have identified residues in the mRNA-protein interface that appear to be critical for stop codon recognition. Especially interesting is a conserved histidine in all bacterial class I release factors that forms a stacking interaction with the second base of the stop codon. Here we analyzed the functional significance of this conserved histidine (position 197 in Escherichia coli) of RF1 by point mutagenesis to alanine. Equilibrium binding studies and transient-state kinetic analysis have shown that the histidine is essential for binding with high affinity to the ribosome. Furthermore, analysis of the binding data indicates a conformational change within the RF1·ribosome complex that results in a more tightly bound state. The rate of peptidyl-tRNA hydrolysis was also reduced significantly, more than the binding data would suggest, implying a defect in the orientation of the GGQ domain without the histidine residue.
Field
Andrew
A
Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0314, USA.
Hetrick
Byron
B
Mathew
Merrill
M
Joseph
Simpson
S
D001665:Binding Sites; D029968:Escherichia coli Proteins; D006639:Histidine; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012270:Ribosomes / Q000378:metabolism
D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D010455:Peptides; D006639:Histidine