Functional specificity of amino acid at position 246 in the tRNA mimicry domain of bacterial release factor 2.
The termination of protein synthesis in bacteria requires codon-specific polypeptide release factors RF-1 (UAG/UAA specific) and RF-2 (UGA/UAA specific). We have proposed that release factors mimic tRNA and recognize the stop codon for polypeptide release (Nakamura et al (1996) Cell 87, 147-150). In contrast to the textbook view, genetic experiments have indicated that Escherichia coli RF-2 terminates translation very weakly at UAA while Salmonella RF-2 decodes this signal efficiently. Moreover, an excess of E coli RF-2 was toxic to cells while an excess of Salmonella RF-2 was not. These two RF-2 proteins are identical except for 16 out of 365 amino acids. Fragment swap experiments and site-directed mutagenesis revealed that a residue at position 246 is solely responsible for these two phenotypes. Upon substituting Ala (equivalent to Salmonella RF-2) for Thr-246 of E coli RF-2, the protein acquired increased release activity for UAA as well as for UGA. These results led us to conclude that E coli RF-2 activity is potentially weak and that the amino acid at position 246 plays a crucial role, not for codon discrimination, but for stop codon recognition or polypeptide release, presumably constituting an essential moiety of tRNA mimicry or interacting with peptidyltransferase centers of the ribosome.
Uno
M
M
Department of Tumor Biology, University of Tokyo, Japan.
Ito
K
K
Nakamura
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D010454:Peptide Termination Factors; D011994:Recombinant Proteins; C041460:peptide chain termination release factor 2; D012343:RNA, Transfer