RESUMO
The Streptomyces coelicolor genome sequence was searched for open reading frames (ORFs) similar to Escherichia coli poly(A) polymerase I, revealing an ORF with 36% amino acid sequence identity to that protein. Mycelial extracts prepared from S. coelicolor cultures incorporated radioactive ATP into an acid-insoluble form, and some of the products of this incorporation had the properties expected of poly(A). [3H]-uridine and [3H]-adenosine were used to label the RNA in S. coelicolor cultures of different ages, and total RNA was fractionated by oligo dT cellulose chromatography. Approximately 3% of the total uridine-labelled RNA and 11% of the adenosine-labelled RNA were retained by the oligo dT cellulose columns. Enzymatic digestion of the retained RNA supported the conclusion that a significant fraction of the adenosine label was present in 3'-poly(A) chains. Measurement of poly(A) tail lengths by end labelling of total RNA and RNase digestion revealed a maximum length of approximately 18 residues. Radioactive cDNA prepared from the RNA fraction retained by oligo dT cellulose hybridized to the 16S and 23S genes from a streptomycete ribosomal RNA operon but not to the 5S gene. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the presence of mRNAs in the RNA fraction retained by oligo dT cellulose.
Assuntos
Proteínas de Escherichia coli , Polinucleotídeo Adenililtransferase/genética , Polinucleotídeo Adenililtransferase/metabolismo , RNA Bacteriano/metabolismo , Streptomyces/metabolismo , Sequência de Aminoácidos , Southern Blotting , Dados de Sequência Molecular , Óperon , Poli A/química , Poli A/metabolismo , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Streptomyces/genéticaRESUMO
We report here the mapping of the transcriptional start point and identification of the promoter for the sigE gene of Streptomyces antibioticus. Sequence analysis revealed a conserved genetic organization of five genes encompassing sigE in S. antibioticus and S. coelicolor. Upstream of sigE a number of direct repeats, while conserved in both species, are arranged differently. Gel shift analysis demonstrated binding of a component of both S. antibioticus and S. coelicolor crude protein extracts to a 30 bp sequence encompassing one repeat, the A-rich box. Deletion analysis in promoter probes showed that maximal activity of the S. antibioticus promoter depends upon the presence of the sequence surrounding the A-rich box, as well as the region further upstream carrying other direct repeats.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , Fator sigma/genética , Streptomyces antibioticus/genética , Sequência de Bases , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The ImmC region of the temperate bacteriophage P1 contains c1, a gene that codes for a repressor of lytic growth. Located in the region upstream of c1 are four open reading frames capable of coding for low-molecular-weight proteins. The efficiency of lysogeny by P1+Cm was found to be reduced by almost 10(5)-fold when the host cells carry this region of ImmC on a multicopy plasmid. The sequences responsible for interfering with lysogen formation were localized to one of the small open reading frames (orf4) within ImmC. Insertions and deletions within orf4 suppress the virulent phenotype of P1virC mutants when introduced into the phage by recombination. These virC-suppressed mutant phage were found to be incapable of lytic growth unless the product of orf4 is provided in trans. The presence of orf4 was observed to interfere with repression by the c1 protein of ImmC-encoded promoters fused to lacZ. For this reason, we suggest that orf4 corresponds to coi, a gene previously proposed to code for an inactivator of c1-mediated repression.
