Role of Cyclic Adenosine 3′,5′-Monophosphate and the Cyclic Adenosine 3′,5′-Monophosphate Receptor Protein in the Initiation of lac Transcription

Abstract

Abstract Optimal transcription of the lac operon in a defined purified in vitro system is dependent upon the concentrations of cyclic adenosine 3',5'-monophosphate (cyclic AMP), the cyclic AMP-binding protein (CRP) and RNA polymerase. The concentration of CRP required for maximal lac transcription varies proportionately with the concentration of lac DNA used as template. Maximal lac transcription is obtained with less CRP when lac ps DNA is used as template than with wild type lac DNA. Both in vivo and in vitro the lac ps mutation appears to result in a more efficient lac promoter. The formation of a preinitiation complex which will synthesize lac mRNA upon addition of nucleoside triphosphates requires preincubation of λh80dlac DNA, RNA polymerase, CRP, and cyclic AMP. The formation of this preinitiation complex is both time and temperature dependent. A lag period of about 2 min is observed between the binding of RNA polymerase to lac DNA and the appearance of detectable lac specific hybridizable RNA. This lag period is possibly due to a slow transition occurring between the formation of the lac preinitiation complex and the onset of extensive polymerization. Our experiments provide further evidence that CRP and cyclic AMP act on lac DNA to stimulate an early step in the initiation of lac transcription by RNA polymerase.