Abstract
Abstract Glycerol added to a transcription system containing λgal DNA and Escherichia coli RNA polymerase holoenzyme stimulates total RNA synthesis and replaces the requirement for cyclic adenosine 3' : 5'-monophosphate (cAMP) and cAMP receptor (CRP) for promotion of gal RNA synthesis. The stimulatory effect which is proportional to the concentration of glycerol up to 20% (v/v) occurs at the level of preinitiation complex formation. In stimulating gal transcription, glycerol appears to act at or close to the same site as cAMP and CRP because: (a) glycerol has little effect on gal transcription at saturating levels of cAMP and CRP; (b) although glycerol stimulates transcription from λgal DNA bearing revertible gal promoter mutations, transcription from DNA containing a gal promoter deletion is not stimulated; (c) glycerol stimulation of gal promoter mutants is inhibited by cAMP and CRP; and (d) with either glycerol or cAMP and CRP, transcription of the promoter-proximal galE region precedes transcription of the galKT region. The effect of glycerol on λ early transcription is to stimulate early r-strand RNA synthesis but not early l-strand RNA synthesis. However, if the DNA template contains a defective λsex promoter, the decreased levels of early l-strand RNA are restored to normal by glycerol. Ethylene glycol, dimethylsulfoxide, sucrose, and 1,3-propanediol, all of which lower the Tm of DNA, also stimulate both total RNA and gal RNA synthesis. Furthermore, the glycerol-promoted formation of the gal preinitiation complex is strongly dependent on the preincubation temperature. At lower temperatures, complex formation requires increased glycerol or decreased KCl concentrations. These findings suggest that glycerol may act by melting or changing the conformation of the DNA promoter regions.
Highlights
If glycerol stimulated the binding of RNA polymerase to the gal promoter in the absence of CAMP and CAMP receptor (CRP), DNA from bacteriophage Xga1326 which is deleted for the gal promoter, should not serve as a template for gal RNA synthesis
6.90 synthesis of gal RNA in the presence of cALlI’ and CRP, indicating that they are defective in gal transcription
When this transcription is reduced by the defective promoter mutations, sesl and sex3, activity is restored to normal by glycerol; and (c) for E. coli gal transcription, glycerol can replace CAMP and CRP and will restore gal RNA synthesis to normal, when defective promoter mutations are present
Summary
DNA and Escherichia coli RNA polymerase holoenzyme stimulates total RNA synthesis and replaces the requirement for cyclic adenosine 3’:5’-monophosphate (CAMP) and CAMP receptor (CRP) for promotion of gal RNA synthesis. Complex formation requires increased glycerol or decreased KC1 concentrations These findings suggest that glycerol may act by melting or changing the conformation of the DNA promoter regions. X rc~prc~ssor csc>rts nrgativc control by binding to the X operators 0, and OH and iutrrfcring with complex formation arid possibly c+mgntion as well [7, 8] Another protein factor, rko, is probably illvolvc,cl in the termination of both gal and X transcription [6, 9]. We observedthat the addition of glycerol to a reaction mixture containing Xgal DNA and RNA polymerasestimulated total RNA synthesis (Fig. 2). When usedasa template for transcription, RNA polymerase by itself will transcribe certain phagesequencebsut will not tran-
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