A novel mutation in ribosomal protein S4 that affects the function of a mutated RF1.
Release factors (RF) 1 and 2 trigger the hydrolysis of the peptide from the peptidyl-tRNA during translation termination. RF1 binds to the ribosome in response to the stop codons UAG and UAA, whereas RF2 recognizes UAA and UGA. RF1 and RF2 have been shown to bind to several ribosomal proteins. To study this interaction in vivo, prfA1, a mutant form of RF1 has been used. A strain with the prfA1 mutation is temperature sensitive (Ts) for growth at 42 degrees C and shows an increased misreading of UAG and UAA. In this work we show that a point mutation in ribosomal protein S4 can, on the one hand, make the RF1 mutant strain Ts(+); on the other hand, this mutation increases the misreading of UAG, but not UAA, caused by prfA1. The S4 mutant allele, rpsD101, is a missense mutation (Tyr51 to Asp), which makes the cell cold sensitive. The behaviour of rpsD101 was compared to the well-studied S4 alleles rpsD12, rpsD14, and rpsD16. These three mutations all confer both a Ts (44 degrees C) phenotype and show a ribosomal ambiguity phenotype, which rpsD101 does not. The three alleles were sequenced and shown to be truncations of the S4 protein. None of the three mutations could compensate for the Ts phenotype caused by the prfA1 mutation. Hence, rpsD101 differs in all studied characteristics from the three above mentioned S4 mutants. Because rpsD101 can compensate for the Ts phenotype caused by prfA1 but enhances the misreading of UAG and not UAA, we suggest that S4 influences the interaction of RF1 with the decoding center of the ribosome and that the Ts phenotype is not a consequence of increased readthrough.
Dahlgren
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A
Department of Microbiology, Stockholm University, 106 91, Stockholm, Sweden.
Rydén-Aulin
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D002876:Chromosomes, Bacterial / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005838:Genotype; D020125:Mutation, Missense; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D012269:Ribosomal Proteins; C489005:prfA protein, E coli; C489006:prfB protein, E coli; C031798:ribosomal protein S4; D001616:beta-Galactosidase