Abstract
The effects of Escherichia coli RNA polymerase and its subassemblies and subunits on the in vitro synthesis of beta subunit directed by DNA from a lambda transducing phage lambdadrif+-6 were investigated. This phage carries the structural gene (rpoB) for beta subunit as well as the genes for EF (translation elongation factor)-Tu, some ribosomal proteins, and stable RNAs of the E. coli chromosome. Among the RNA polymerase proteins examined, the two oligomers, holoenzyme and alpha2beta complex, repressed the synthesis of only the beta subunit but not of other proteins encoded by the phage DNA. The results indicate that the expression of at least the betabeta' (rpoBC) operon is under autogenous regulation, in which both holoenzyme and alpha2beta complex function as regulatory molecules with repressor activity.
Highlights
Proteins Synthesized in Vitro by Xdrif+ DNA-A number of transducing phages carrying the genes coding for the RNA
The phage hdrifc-6 was isolated by Ikeuchi et al [11] and shown to carry 0.54% of the E. coli chromosome between bfe and rif(rpoB) including the structural gene for fl subunit but not for p’ subunit (TOG’) [9,23]
Based on the analyses of gene products encoded by another rif-transducing X phage [24,25], it is expected that the structural genes for EF-Tu, ribosomal proteins Ll, L8, LlO (r&J), Lll, L7/L12, and some stable RNAs are located in the region integrated into the transducing phage
Summary
Preparation of Xdrif+-6 DNA-A phage lysate containing hcZ857driF-6(designated as hdrif+ in this report) and XcZ’ was prepared by heat induction of a double lysogen E. coli KY3372 (F-thi thr Zeu metB rha str tsx XH (XcZ+) (XcZ857-drift-6) supplied by Dr.T. Preparation of Xdrif+-6 DNA-A phage lysate containing hcZ857driF-6. (designated as hdrif+ in this report) and XcZ’ was prepared by heat induction of a double lysogen E. coli KY3372 Thi thr Zeu metB rha str tsx XH (XcZ+) (XcZ857-drift-6) supplied by Dr. T. Yura), followed by addition of Mitomycin C [11]. Phage particles were precipitated with polyethylene glycol, purified by CsCl step gradient centrifugation, and separated into helper and transducing phage by equilibrium centrifugation in CsCl gradient. DNA was extracted from the purified phage particles with phenol and dialyzed against 10 mM Tris’ acetate, pH 8.0, before use
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