RESUMO
The transcriptional organization of the erythromycin biosynthetic gene (ery) cluster of Saccharopolyspora erythraea has been examined by a variety of methods, including S1 nuclease protection assays, Northern blotting, Western blotting, and bioconversion analysis of erythromycin intermediates. The analysis was facilitated by the construction of novel mutants containing a S. erythraea transcriptional terminator within the eryAI, eryAIII, eryBIII, eryBIV, eryBV, eryBVI, eryCIV, and eryCVI genes and additionally by an eryAI -10 promoter mutant. All mutant strains demonstrated polar effects on the transcription of downstream ery biosynthetic genes. Our results demonstrate that the ery gene cluster contains four major polycistronic transcriptional units, the largest one extending approximately 35 kb from eryAI to eryG. Two overlapping polycistronic transcripts extending from eryBIV to eryBVII were identified. In addition, seven ery cluster promoter transcription start sites, one each beginning at eryAI, eryBI, eryBIII, eryBVI, and eryK and two beginning at eryBIV, were determined.
Assuntos
Eritromicina/biossíntese , Família Multigênica , Saccharopolyspora/genética , Transcrição Gênica , Sequência de Bases , Northern Blotting , Western Blotting , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , RNA Bacteriano/genética , RNA Ribossômico/genética , Saccharopolyspora/metabolismo , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Regiões Terminadoras Genéticas , Óperon de RNArRESUMO
The prs gene, encoding phosphoribosylpyrophosphate synthetase, is preceded by a leader, which is 302 bp long in Escherichia coli and 417 bp in Salmonella typhimurium. A potential open reading frame (ORF) extends across the prs promoter and into the leader. The region between the prs coding region and an upstream gene (hemA) in E. coli and S. typhimurium was cloned, sequenced and shown to encode two ORFs of unknown function. ORF 1 encodes a 23 kDa protein and ORF 2 a 31 kDa protein, as observed by denaturing PAGE of extracts of cells bearing plasmids encoding the ORFs. Both ORFs are transcribed in the same direction as the prs gene with ORF 2 extending into the prs leader. Northern blot analysis showed that the prs message in E. coli was on 1.3 and 2.7 kb transcripts. The shorter transcript encoded the prs gene only, while the longer transcript also encoded the two ORFs. Thus, the prs gene is transcribed from two promoters, the first promoter (P1) originating upstream of ORF 1, and expressing the prs gene in a tricistronic operon and a second promoter (P2), located within the ORF 2 coding frame, which transcribes the prs gene only. The transcripts encoding prs only were 20 times as abundant as the tricistronic transcripts under all conditions examined. This was the case whether cells containing plasmid-encoded or only chromosomally encoded copies of the hemA-prs region were probed for these transcripts. Derepression of the prs gene upon pyrimidine starvation was shown to be due to an increase in the amount of message originating from the promoter P2.
Assuntos
Aldeído Oxirredutases/genética , Proteínas de Bactérias/genética , Escherichia coli/genética , Fases de Leitura Aberta , Ribose-Fosfato Pirofosfoquinase/genética , Salmonella typhimurium/genética , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Cromossomos Bacterianos , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Pirimidinas/metabolismo , Sondas RNA , RNA Bacteriano/genética , RNA Mensageiro/genética , Salmonella typhimurium/metabolismoRESUMO
The Salmonella typhimurium prsB mutation was previously mapped at 45 min on the chromosome, and a prsB strain was reported to produce undetectable levels of phosphoribosylpyrophosphate (PRPP) synthetase activity and very low levels of immunologically cross-reactive protein in vitro (N.K. Pandey and R.L. Switzer, J. Gen. Microbiol, 128:1863-1871, 1982). We have shown by P22-mediated transduction that the prsB gene is actually an allele of prsA, the structural gene for PRPP synthetase, which maps at 35 min. The prsB (renamed prs-100) mutant produces about 20% of the activity and 100% of the cross-reactive material of wild-type strains. prs-100 mutant strains are temperature sensitive, as is the mutant PRPP synthetase in vitro. The prs-100 mutation is a C-to-T transition which results in replacement of Arg-78 in the mature wild-type enzyme by Cys. The mutant PRPP synthetase was purified to greater than 98% purity. It possessed elevated Michaelis constants for both ATP and ribose-5-phosphate, a reduced maximal velocity, and reduced sensitivity to the allosteric inhibitor ADP. The mutant enzyme had altered physical properties and was susceptible to specific cleavage at the Arg-101-to-Ser-102 bond in vivo. It appears that the mutation alters the enzyme's kinetic properties through substantial structural alterations rather than by specific perturbation of substrate binding or catalysis.
