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Y V Brun

Publications and source records attributed to Y V Brun.

6 recordsLinked to original sources

A temporally controlled sigma-factor is required for polar morphogenesis and normal cell division in Caulobacter.

The transcription of many spatially and temporally controlled flagellar structural genes in Caulobacter requires the RNA polymerase sigma 54 subunit. Like flagellar biogenesis, stalk formation is an asymmetric polar morphogenesis that occurs once each cell cycle in response to internal cell cycle signals. We have isolated the sigma 54 gene (rpoN) and describe here a novel role for this alternative sigma-factor in cell differentiation: It is required for the biogenesis of both polar structures, and the disruption of the rpoN gene results in aberrant cell division. Surprisingly, the transcription of rpoN is temporally regulated during the cell cycle; it increases 10-fold commensurate with stalk formation and just before the onset of flagellar gene expression. These results suggest that sigma 54 abundance responds to cell cycle cues and is involved in the global timing of the central events of Caulobacter development, whereas the transcriptional activators of sigma 54-dependent promoters are responsible for the refined control of the expression of individual or small groups of genes required for each specific event.

Amino Acid Sequence

Large scale sequencing projects using rapidly prepared double-stranded plasmid DNA.

We have developed a simple rapid plasmid DNA mini-preparation method which yields DNA of sufficient quality to be used in large scale sequencing projects. The method, which is a modification of the alkaline method of Birnboim and Doly (1979), requires less than two hours. We have eliminated the use of organic extractions, RNase digestion and alkaline denaturation of the DNA for annealing of the primer. The proportion of supercoiled plasmid DNA obtained is close to 100%. Greater than 80% of the clones yield at least 500 bp of sequence information per primer. The sequencing reactions from these double-stranded templates can be done on both strands using the universal and reverse sequence primers with the usual two reactions per primer, one to read close to the primer and one to read far from it. Thus, each clone yields at least 1 kb of sequence information. The preparation of the templates and the sequencing reactions can be done in less than three hours so that the sequencing gel can be run the same day.

Base Sequence

Precise mapping and comparison of two evolutionarily related regions of the Escherichia coli K-12 chromosome. Evolution of valU and lysT from an ancestral tRNA operon.

Two tRNA operons have been found near the gltX gene encoding the glutamyl-tRNA synthetase of Escherichia coli K-12. The alaW operon previously undetected from genetic data and containing two identical tRNA(GGCAla) genes is 800 base-pairs downstream from the gltX terminator and is transcribed from the same strand. The valU operon containing genes for three identical tRNA(UACVal) and one tRNA(UUULys) (the wild-type allele of supN), is adjacent to gltX and is transcribed from the opposite strand. Five open reading frames were also found in this region encoding putative polypeptides of 62, 105, 130, 167 and 294 amino acid residues. ORF294 is a new member of the lysR family of bacterial transcriptional activators. The possibility that this is the xapR gene is discussed. Comparison of the physical and linkage maps of the E. coli chromosome in the 52 minute region has permitted precise mapping of most of the 18 genes in this region with the order nupC-glk- less than (alaW beta-ala W alpha)-1 kb- less than gltX-0.3 kb-(valU alpha-valU beta-valU gamma-lysV = supN) greater than xapR-xapA- less than lig-1 kb-cysK greater than -0.4 kb-ptsH greater than -0.05 kb-pstI greater than -0.05 kb-crr greater than -cysM-cysA in the clockwise order (greater than and less than indicate the direction of transcription; kb, 10(3) bases). The last two genes of valU (52 min) and lysT (16.5 min) are arranged in a similar fashion and a highly conserved region has been found in both operons. This suggests that the valU and lysT operons probably arose by a duplication of an ancestral tRNA operon. This is the first example of what may be two different tRNA operons from the same organism evolving from an ancestral tRNA gene. Comparison of the 16 and 52 minute regions of the E. coli K-12 chromosome suggests that these two regions could share a common ancestor.

Amino Acid Sequence

Closely spaced and divergent promoters for an aminoacyl-tRNA synthetase gene and a tRNA operon in Escherichia coli. Transcriptional and post-transcriptional regulation of gltX, valU and alaW.

The transcription of the gltX gene encoding the glutamyl-tRNA synthetase and of the adjacent valU and alaW tRNA operons of Escherichia coli K-12 has been studied. The alaW operon containing two tRNA(GGCAla) genes, is 800 base-pairs downstream from the gltX terminator and is transcribed from the same strand. The valU operon, containing three tRNA(UACVal) and one tRNA(UUULys) (the wild-type allele of supN) genes, is adjacent to gltX and is transcribed from the opposite strand. Its only promoter is upstream from the gltX promoters. The gltX gene transcript is monocistronic and its transcription initiates at three promoters, P1, P2 and P3. The transcripts from one or more of these promoters are processed by RNase E to generate two major species of gltX mRNA, which are stable and whose relative abundance varies with growth conditions. The stability of gltX mRNA decreases in an RNase E- strain and its level increases with growth rate about three times more than that of the glutamyl-tRNA synthetase. The 5' region of these mRNAs can adopt a stable secondary structure (close to the ribosome binding site) that is similar to the anticodon and part of the dihydroU stems and loops of tRNA(Glu), and which might be involved in translational regulation of GluRS synthesis. The gltX and valU promoters share the same AT-rich and bent upstream region, whose position coincides with the position of the upstream activating sequences of tRNA and rRNA promoters to which they are similar. This suggests that gltX and valU share transcriptional regulatory mechanisms.

Amino Acyl-tRNA Synthetases

Overproduction and domain structure of the glutamyl-tRNA synthetase of Escherichia coli.

The charging of glutamate on tRNA(Glu) is catalyzed by glutamyl-tRNA synthetase, a monomer of 53.8 kilodaltons in Escherichia coli. To obtain the large amounts of enzyme necessary for the identification of structural domains, we have inserted the structural gene gltX in the conditional runaway-replication plasmid pOU61, which led to a 350-fold overproduction of glutamyl-tRNA synthetase. Partial proteolysis of this enzyme revealed the existence of preferential sites of attack that, according to their N-terminal sequences, delimit regions of 12.9, 2.3, 12.1, and 26.5 kilodaltons from the N- to C-terminal of the enzyme. Their sizes suggest that the 2.3-kilodalton fragment is a hinge structure, and that those of 12.9, 12.1, and 26.5 kilodaltons are domain structures. The 12.9-kilodalton domain of the glutamyl-tRNA synthetase of E. coli is the only long region of this enzyme displaying a good amino acid sequence similarity with the glutaminyl-tRNA synthetase of Escherichia coli.

Amino Acid Sequence