Studies with cyclic adenosine monophosphate receptor and stimulation of in vitro transcription of the Gal operon.

To elucidate further how cyclic adenosine 3’ : S’-monophosphate (cyclic AMP) and the cyclic AMP receptor are able to stimulate gal mRNA synthesis we have utilized an antiserum specific for cyclic AMP receptor and also cyclic GMP as inhibitors of cyclic AMP and cyclic AMP receptor action. When cyclic AMP, cyclic AMP receptor, DNA, and RNA polymerase are incubated together a rifampicin-resistant preinitiation complex is formed and, upon the addition of missing ribonucleoside triphosphates or Mg2+, gal mRNA synthesis occurs. If the preinitiation complex is formed in the absence of nucleoside triphosphates the antiserum is able to dissociate the complex. If the complex is formed in the presence of nucleoside triphosphates (no Mgz+), it is converted to a more stable form which is not reversed by the addition of antiserum. Cyclic GMP can prevent the formation of the preinitiation complex. However, we find that once the preinltiation complex has been formed cyclic GMP does not inhibit cyclic AMP receptor-cyclic AMP-dependent transcription, suggesting that RNA polymerase stabilizes cyclic AMP receptor-cyclic AMP binding to the gal promoter region.