RESUMO
Specific cleavage of mRNAs by RNase III has been shown to control the expression of several Escherichia coli genes. We show here that the expression of gene 19 of the conjugative resistance plasmid R1 is controlled in its expression by the same endoribonuclease. In vivo studies revealed that a DNA fragment of 150 nucleotides including a perfect 22 nucleotide inverted repeat in the gene 19 coding region is responsible for the low expression of the gene both at the protein and the RNA levels. By using a translational gene 19-lacZ fusion in isogenic RNase III+ and RNase III- strains we could identify RNase III as the key element in the down-regulation of gene 19 expression. The sequencing of in vitro generated and RNase III-digested transcripts confirmed the in vivo studies and revealed the exact positions of the RNase III cleavage sites within the coding part of the gene 19 transcript. The in vitro determined RNase III cleavage of gene 19 mRNA was confirmed by in vivo primer extension analysis. Finally, we could show that an exchange of three nucleotides within the RNase III recognition site abolished RNase III cleavage in vitro.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Endorribonucleases/fisiologia , Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Fatores R/genética , Processamento Pós-Transcricional do RNA , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Conjugação Genética , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/biossíntese , Sequências Repetitivas de Ácido Nucleico , Ribonuclease IIIRESUMO
The nucleotide sequences of the enterotoxin plasmid P307 transfer genes traM, finP, traJ, traY, and gene 19 were determined. Gene 19 is highly conserved; its product is very similar to that coded by the F and R1 plasmids. The TraM protein is similar in P307 and in F; the R1 sequence shows differences in the 40 N-terminal amino acids. The traJ product is very different in P307, F, and R1. The traY gene from P307, which in F is almost twice as long, is similar in size to that from R1. The finP RNA shows a high degree of homology with that from R1 and F, except for the two loop regions where base changes were observed. The genes coding for proteins, except traY, could be expressed in minicell- and T7 promoter-driven expression systems, whereas traJ and gene 19 could be expressed only in the latter system.
Assuntos
Enterotoxinas/genética , Plasmídeos , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , DNA Bacteriano/genética , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
The DNA fragment carrying the oriT region from the enterotoxin plasmid P307 was isolated and its polynucleotide sequence was determined. Using Southern hybridization assays with a synthetic oligonucleotide probe, the oriT region was identified on a 7.9-kb EcoRI fragment from P307. By ligating the fragment with the cloning vector pUC119, plasmid pAG10 was obtained. The physical map of the insert was determined and oriT was located on a 540-bp BglII/SalI fragment. After this fragment was subcloned into sequencing phages, the polynucleotide sequence was established. Part of the sequence proved to be almost identical to segments of the oriT regions of the plasmids F and R1; another neighboring region was very different among all three sequences. The polynucleotide sequence proximal to traM is highly similar to that of F but different from that of R1.
