TAILIEUCHUNG - Báo cáo khóa học: Generation and characterization of functional mutants in the translation initiation factor IF1 of Escherichia coli

Three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. No clear function has been assigned to the smallest of these three factors, IF1. Therefore, to investigate the role of this protein in the initi-ation process inEscherichia coliwe have mutated the cor-responding gene infA. Because IF1 is essential for cell viability and no mutant selection has so far been described, the infA gene in a plasmid was mutated by site-directed mutagenesis in a strain with a chromosomalinfA + gene, followedbydeletionof thisinfA + this approach, the six arginine residues of IF1 were altered to leucine or aspartate | Eur. J. Biochem. 271 534-544 2004 FEBS 2004 doi Generation and characterization of functional mutants in the translation initiation factor IF1 of Escherichia coli Victor Croitoru Margarete Bucheli-Witschel Peter Hagg Farhad Abdulkarim and Leif A. Isaksson Department of Microbiology Stockholm University Sweden Three protein factors IF1 IF2 and IF3 are involved in the initiation of translation in prokaryotes. No clear function has been assigned to the smallest of these three factors IF1. Therefore to investigate the role of this protein in the initiation process in Escherichia coli we have mutated the corresponding gene infA. Because IF1 is essential for cell viability and no mutant selection has so far been described the infA gene in a plasmid was mutated by site-directed mutagenesis in a strain with a chromosomal inf A gene followed by deletion of this infA gene. Using this approach the six arginine residues of IF1 were altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. All mutants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable except for an R65D alteration. Differences in growth phenotypes of the mutants were observed in both minimal and rich media. Some of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type infA allele. Several of these recombinants showed reduced growth rate and a partial coldsensitive phenotype. This paper presents a collection of IF1 mutants designed for in vivo and in vitro studies on the function of IF1. Keywords initiation factor IF1 mutagenesis translation initiation. Translation initiation is the first step in protein synthesis and comprises several steps a the 70S ribosome dissociates into 30S and 50S subunits b a 30S initiation complex is formed in which initiator fMet-tRNA interacts with the .

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