Frédéric Dardel

Molecular recognition governing the initiation of translation in Escherichia coli. A review

Selection of the proper start codon for the synthesis of a polypeptide by the Escherichia coli translation initiation apparatus involves several macromolecular components. These macromolecules interact in a specific and concerted manner to yield the translation initiation complex. This review focuses on recent data concerning the properties of the initiator tRNA and of enzymes and factors involved in the translation initiation process. The three initiation factors, as well as methionyl-tRNA synthetase and methionyl-tRNA(f)Met formyltransferase are described. In addition, the tRNA recognition properties of EF-Tu and peptidyl-tRNA hydrolase are considered. Finally, peptide deformylase and methionine aminopeptidase, which catalyze the amino terminal maturation of nascent polypeptides, can also be associated to the translation initiation process.

Genetic engineering of methionyl-tRNA synthetase: in vitro regeneration of an active synthetase by proteolytic cleavage of a methionyl-tRNA synthetase-β-galactosidase chimeric protein

The construction of a family of plasmids carrying derivatives of metG, the gene for E. coli methionyl-tRNA synthetase, is described. These plasmids allow expression of native or truncated forms of the enzyme and easy purification of the products. To facilitate the characterization of modified enzymes with very low catalytic activity, a specialized vector was constructed, in which metG was fused in frame with lacZ, the gene for beta-galactosidase. This plasmid expresses a methionyl-tRNA synthetase-beta-galactosidase chimeric protein, which is shown to carry the activities of both enzymes. This hybrid can be purified in a single step of affinity chromatography for beta-galactosidase. The methionyl-tRNA synthetase moiety can be regenerated by mild proteolysis, thus providing a simple method for purifying and studying mutated proteins.

Transcription and regulation of expression of the Escherichia coli methionyl-tRNA synthetase gene

SummaryThe DNA sequence and transcriptional organization around the Escherichia coli methionyl-tRNA synthetase gene, metG, were resolved. This gene can be transcribed in vivo and in vitro from two distinct promoters separated by 510 nucleotides. The upstream promoter is located within the coding sequence of a divergent gene expressing a protein of Mr 39 kDa of unknown function. Transcription originating from this upstream promoter is attenuated by a Rho-independent terminator before entering the structural gene. This leader RNA contains several potentially stable secondary structures, one of which shows striking similarity to tRNAMet, but no methionine-rich coding sequence. The regulation of metG expression was investigated by means of fusions to the lacZ gene. Transcription of a metG: : lacZ fusion is induced in a metG mutant and, reciprocally, repression is observed in a methionyl-tRNA synthetase overproducing strain. A model of metG expression control is proposed.

THE N-TERMINAL HALF OF INITIATION FACTOR IF3 IS FOLDED AS A STABLE INDEPENDENT DOMAIN

Initiation factor IF3 plays an essential role in the initiation of protein translation by binding to the 30S ribosomal subunit and selecting a proper tRNA(fMet)/initiation codon complex. The domain structure of IF3 from Escherichia coli has been investigated by limited proteolysis followed by mass spectrometry and protein sequencing of the resulting peptides. This analysis revealed a highly segmented structure with two independent domains connected by a charged linker peptide, highly susceptible to proteolytic cleavage. The N-terminal domain is very stable and comparison of its 2-D NMR spectrum with that of intact IF3 revealed that it retains its three-dimensional fold.

Methionyl-tRNA synthetase from E coli - A review

Methionyl-tRNA synthetase (MetRS) from E coli is a dimer composed of 2 identical subunits of Mr 76 kDa. A fully active monomeric fragment (64 kDa) could be obtained by mild proteolysis of the native dimer. Earlier studies reviewed in Blanquet et al (1979) have compared the catalytic mechanisms of native and trypsin-modified MetRS. Moreover, the truncated form of the enzyme was crystallized and its 3-D structure solved at low resolution. In the last few years, the availability of the corresponding metG gene has facilitated the development of studies using affinity labelling and site-directed mutagenesis techniques. In parallel, the 3-D structure has been solved at a resolution of 2.5 A. These convergent approaches have allowed significant progress in the understanding of the structure-function relationships of this enzyme, and, in particular, of the rules governing the recognition of tRNA.

DNAid: a Macintosh full screen editor featuring a built-in regular expression interpreter for the search of specific patterns in biological sequences using finite state automata

DNAid is a full screen and multi-window sequence editor designed as an aid for the easy manipulation of DNA and protein sequences on Macintosh computers. In addition to the classical editing capabilities, powerful analysis and search functions are available from within the editor. Restriction mapping, translation and alignment of homologous sequences are supported by DNAid, furthermore a pattern matching language is included which allows searches for user-defined strict or fuzzy signals within biological sequences. Patterns are translated into finite state automata which allow very efficient searches.

Open reading frames in the control regions of the phenylalanyl-tRNA synthetase operon of E. coli

The pheST operon codes for the two subunits of phenylalanyl-tRNA synthetase and it expression is controlled by attenuation in a way similar to many amino acid biosynthetic operons. The nucleotide sequence of the control regions of the operon indicates the presence of several open reading frames besides that of the leader peptide. One of these open reading frames, called the alternative leader peptide, starts at about the same place as the leader peptide and ends after the terminator of the attenuator. Another open reading frame, called the terminator peptide, starts after the terminator and covers about half the distance to pheS, the first structural gene of the operon. The present report shows that, in fact, the only open reading frame to be translated efficiently is the leader peptide itself. The alternative leader peptide and the terminator peptide are both translated at a negligible rate.