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The antibiotic kirromycin inhibits protein synthesis by binding to EF-Tu and preventing its release from the ribosome after GTP hydrolysis. We have isolated and sequenced a collection of kirromycin resistant tuf mutations and identified thirteen single amino acid substitutions at seven different sites in EF-Tu. These have been mapped onto the 3D structures of EF-Tu'GTP and EF-Tu.GDP. In the active GTP form of EF-Tu the mutations cluster on each side of the interface between domains I and III. We propose that this domain interface is the binding site for kirromycin.

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 infA + 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 initiation codon of an mRNA bound to the 30S ribosomal subunit; (c) finally, the 50S subunit joins to form the 70S initiation complex that then enters the elongation phase of translation. These reactions, in particular the formation of the 30S initiation complex, are promoted by the three initiation factors IF1, IF2 and IF3 [1]. In contrast to IF2 and IF3, the role of IF1 is uncertain and so far no clear specific function has been assigned to this protein [2,3] even though IF1 is essential for cell viability in Escherichia coli [4]. Apart from this latter in vivo study most other available data on IF1 have been gathered by in vitro investigations (for an overview see Sette et al.IF1 is a small protein consisting of 71 amino acids [6] and its structure contains an oligomer-binding motif which binds to oligonucleotides or oligosaccharides [5]. The oligomer-binding motif is also present in other nucleicacid binding proteins such as cold shock protein A or aspartyl-tRNA-synthetase. For several of these proteins it has been demonstrated that basic and aromatic amino acids are involved in the binding to RNA or DNA. Moreover, previous NMR studies have suggested that the arginine and lysine residues of IF1 are involved in binding to the 30S ribosomal subunit [7]. IF1 binds to the 30S subunit in a 1 : 1 ratio [8,9] and this interaction is enhanced in the presence of IF2 and IF3. This effe...

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