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Functional sites of interaction between release factor RF1 and the ribosome

Abstract

Translational release factors decipher stop codons in mRNA and activate hydrolysis of peptidyl-tRNA in the ribosome during translation termination. The mechanisms of these fundamental processes are unknown. Here we have mapped the interaction of bacterial release factor RF1 with the ribosome by directed hydroxyl radical probing. These experiments identified conserved domains of RF1 that interact with the decoding site of the 30S ribosomal subunit and the peptidyl transferase site of the 50S ribosomal subunit. RF1 interacts with a binding pocket formed between the ribosomal subunits that is also the interaction surface of elongation factor EF-G and aminoacyl-tRNA bound to the A site. These results provide a basis for understanding the mechanism of stop codon recognition coupled to hydrolysis of peptidyl-tRNA, mediated by a protein release factor.

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Figure 1: Sites of single Cys mutations engineered into RF1, translational termination complex, and peptidyl-tRNA hydrolysis assay.
Figure 2: Directed hydroxyl radical probing of the interaction of RF1 with the ribosome.
Figure 3: Summary of directed hydroxyl radical cleavages obtained from various Fe(II)-tethered RF1 proteins.
Figure 4: RF1, aminoacyl-tRNA, and EF-G interact with an overlapping binding pocket on the ribosome.

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Acknowledgements

We thank M. Uno for constructing the Cys derivative at position 229, R. Green for the plasmid containing T4 gene 32, and K. Fredrick and J. Diener for comments on the manuscript. This work was supported by grants (to Y.N.) from The Ministry of Education, Science, Sports and Culture, Japan, the Human Frontier Science Program and the Basic Research for Innovation Biosciences Program of Bio-oriented Technology Research Advancement Institution (BRAIN), and by grants from the NIH (to H.F.N.) and the W.M. Keck Foundation (to the Center for Molecular Biology of RNA). K.S.W. is an American Cancer Society Postdoctoral Fellow, and K.I. is a BRAIN senior investigator.

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Correspondence to Kevin S. Wilson or Yoshikazu Nakamura.

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Wilson, K., Ito, K., Noller, H. et al. Functional sites of interaction between release factor RF1 and the ribosome. Nat Struct Mol Biol 7, 866–870 (2000). https://doi.org/10.1038/82818

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