RESUMO
Using a gene probe of the Escherichia coli groEL gene, a 1.8-kb HindIII fragment of chromosomal DNA of Bacillus subtilis was cloned. Upstream sequences were isolated as a 3-kb PstI fragment. Sequencing of 2,525 bp revealed two open reading frames in the order groES groEL. Alignment of the GroES and GroEL proteins with those of eight other eubacteria revealed 50 to 65% and 72 to 84% sequence similarity, respectively. Primer extension studies revealed one potential transcription start site preceding the groESL operon (S) which was activated upon temperature upshift. Northern (RNA) analysis led to the detection of two mRNA species of 2.2 and 1.5 kb. RNA dot blot experiments revealed an at least 10-fold increase in the amount of specific mRNA from 0 to 5 min postinduction, remaining at this high level for 10 min and then decreasing. A 9-bp inverted repeat within the 5' leader region of the mRNA might be involved in regulation of the heat shock response. By using PBS1 transduction, the groESL operon was mapped at about 342 degrees.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Choque Térmico/genética , Óperon/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Northern Blotting , Chaperonina 10 , Chaperonina 60 , Clonagem Molecular , Proteínas de Choque Térmico/química , Dados de Sequência Molecular , Plasmídeos/genética , Mapeamento por Restrição , Transcrição Gênica/genéticaRESUMO
By using an internal part of the dnaK gene from Bacillus megaterium as a probe, a 5.2-kb HindIII fragment of chromosomal DNA of Bacillus subtilis was cloned. Downstream sequences were isolated by in vivo chromosome walking. Sequencing of 5,085 bp revealed four open reading frames in the order orf39-grpE-dnaK-dnaJ. orf39 encodes a 39-kDa polypeptide of unknown biological function with no noticeable homology to any other protein within the data bases. Alignment of the GrpE protein with those of three other bacterial species revealed a low overall homology, but a higher homology restricted to two regions which might be involved in interactions with other proteins. Alignment of the DnaK protein with six bacterial DnaK polypeptides revealed that a contiguous region of 24 amino acids is absent from the DnaK proteins of all known gram-positive species. Primer extension studies revealed three potential transcription start sites, two preceding orf39 (S1 and S2) and a third one in front of grpE (S3). S2 and S3 were activated at a high temperature. Northern (RNA) analysis led to the detection of three mRNA species of 4.9, 2.6, and 1.5 kb. RNA dot blot experiments revealed an at-least-fivefold increase in the amount of specific mRNA from 0 to 5 min postinduction and then a rapid decrease. A transcriptional fusion between dnaK and the amyL reporter gene exhibited a slight increase in alpha-amylase activity after heat induction. A 9-bp inverted repeat was detected in front of the coding region of orf39. This inverted repeat is present in a number of other heat shock operons in other microorganisms ranging from cyanobacteria to mycobacteria. The biological property of this inverted repeat as a putative key element in the induction of heat shock genes is discussed. The dnaK locus was mapped at about 223 degrees on the B. subtilis genetic map.
Assuntos
Bacillus subtilis/genética , Proteínas de Choque Térmico/genética , Sequência de Aminoácidos , Bacillus/genética , Bacillus megaterium/genética , Proteínas de Bactérias/genética , Sequência de Bases , Passeio de Cromossomo , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico HSP40 , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/genética , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , alfa-Amilases/genéticaRESUMO
The interaction of 18 different Escherichia coli aminoacyl-tRNA species with elongation factor Tu and GTP has been measured by a fluorescence titration assay under equilibrium conditions. The dissociation constants range from 1.9 +/- 0.2.10(-10) M up to 1020 +/- 250.10(-10) M depending on the nucleotide sequence, secondary structure and the chemical composition of the aminoacyl residue of the particular aminoacyl-tRNA. The 'aminoacyl domain' of tRNA consisting of the single stranded, four-nucleotide-long 3'-terminus, aminoacyl stem of seven base-pairs, T-stem and T-loop contains all elements necessary for binding EF-Tu.GTP. The efficiency of aminoacyl-tRNA interaction with EF-Tu.GTP is modulated by the sequence of this 'aminoacyl domain' and by natural modification of its nucleotide residues. An oligoribonucleotide resembling the aminoacyl stem of E.coli tRNA(Ala) and consisting of a four-membered 3'-end, a stem of seven base-pairs and a loop of six nucleotides was prepared by total chemical synthesis on a polymer support. It can be enzymatically aminoacylated by alanine but does not bind in its aminoacylated form to EF-Tu.GTP.
