Bacteriophage lambda vehicle for the direct cloning of Escherichia coli promoter DNA sequences: feedback regulation of the rplJL-rpoBC operon.
A derivative of bacteriophage lambda, lambda 21, has been constructed and used for the cloning of Escherichia coli DNA fragments carrying promoters. Phage lambda 21 lacks the lac promoter operator and can accept DNA fragments up to 9.8 kilobases in size at a unique HindIII restriction endonuclease site adjacent to lacZ. Recombinant phage that carry promoters are readily identified by their expression of lacZ. Lysogens of these phages in strains harboring a deletion of the chromosomal lac operon are capable of growth on lactose as sole carbon source and can be used to study some of the regulatory signals that act upon the cloned promoter. In principle, lambda 21 can be used to clone any promoter DNA sequence with HindIII termini. PJ, the primary promoter for the rplJL-rpoBC operon, and P beta, a weak promoter for rpoBC, have been cloned in lambda 21. Transcription of lacZ from PJ was found to be subjected to feedback control by ribosomal protein L10 and to a lesser extent by ribosomal protein L7/L12. This suggests a possible L10-binding site near PJ that regulates transcription from that promoter. Lysogens of the phage that carries P beta responded to two regulatory signals: a rho-sensitive termination site preceding rpoBC and induction of beta-galactosidase synthesis by rifampicin. This suggests that P beta is a bona fide promoter for rpoBC.
D001426:Bacterial Proteins / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D009876:Operon; D012269:Ribosomal Proteins / Q000235:genetics*; D012293:Rifampin / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D001616:beta-Galactosidase / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012269:Ribosomal Proteins; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase; D012293:Rifampin