End-product control of expression of branched-chain amino acid biosynthesis genes in Streptomyces coelicolor A3(2): paradoxical relationships between DNA sequence and regulatory phenotype.
The branched-chain protein amino acids isoleucine, valine and leucine can provide precursors for synthesis of complex polyketide secondary metabolites in streptomycetes; therefore the regulation of their own synthesis is of interest. DNA sequences upstream of ilvBNC, ilvD, leuA, leuB, ilvE and leuCD in Streptomyces coelicolor A3(2) have been obtained in this laboratory or as part of the S. coelicolor genome sequencing project. Upstream of ilvB and leuA, typical features of classical attenuator systems can be discerned, in particular hypothetical short ORFs with runs of Ile/Val/Leu and Leu codons, respectively. No such features are apparent upstream of other genes or gene clusters present. All five upstream regions were fused to xylE (encoding catechol dioxygenase, CO) as a reporter gene in the SCP2*-based low-copy-number vector pIJ2839. All wild-type regions showed strong depression of CO activity in the presence of all three branched-chain amino acids whether or not the attenuation features were present. By site-directed mutagenesis, the Ile/Val/Leu and Leu triplets in the putative attenuator peptides for ilvB and leuA were replaced by ones for other amino acids. In the case of ilvB, this had no effect at all; for leuA, the wild-type regulatory phenotype persisted in at least some experiments. It was concluded that (i) an unknown regulatory mechanism must be operating in the ilv/leu system of S. coelicolor A3(2) in place of classical attenuation; and (ii) it is unsafe to infer the functioning of a regulatory mechanism from sequence homologies alone.
Craster
Heather L
HL
School of Biology, University of Leeds, Leeds LS2 9JT, UK1.
Potter
Chris A
CA
School of Biology, University of Leeds, Leeds LS2 9JT, UK1.
Baumberg
Simon
S
School of Biology, University of Leeds, Leeds LS2 9JT, UK1.
D020121:5' Untranslated Regions; D000595:Amino Acid Sequence; D000597:Amino Acids, Branched-Chain / Q000096:biosynthesis* / Q000235:genetics*; D001483:Base Sequence; D050557:Catechol 2,3-Dioxygenase; D049308:Dioxygenases; D015964:Gene Expression Regulation, Bacterial; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D007535:Isomerases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D000597:Amino Acids, Branched-Chain; D012329:RNA, Bacterial; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase; D006836:Hydro-Lyases; C032241:isopropylmalate isomerase; C020052:dihydroxyacid dehydratase; D007535:Isomerases