Cyclic-AMP-dependent switch in initiation of transcription from the two promoters of the Escherichia coli gal operon: identification and assay of 5'-triphosphate ends of mRNA by GTP:RNA guanyltransferase

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Abstract
We have studied the initiation of transcription of the gal operon in Escherichia coli (i) by analyzing the 5'-triphosphate ends and (ii) by measuring the level of promoter-proximal gal mRNA made in vivo. The 5' termini were identified and quantified by capping with GTP:mRNA guanyltransferase, and the mRNA levels were determined by hybridization of pulse-labeled [32P]RNA with a specific DNA probe. Our results conclusively demonstrate the in vivo activities of two promoters, P1 and P2, with separate initiation sites (S1 and S2) as suggested before from in vitro and in vivo experiments (S. Adhya and W. Miller, Nature [London] 279:492-494, 1979; R. E. Musso, R. DiLauro, S. Adhya, and B. de Crombrugghe, Cell 12:847-854, 1977). We have also studied the effect of cyclic AMP (cAMP) on in vivo gal transcription and found that whereas total gal transcription remains largely unchanged, the relative proportions of the S1 and S2 mRNAs are influenced by the level of cAMP in the cell. In strains devoid of cAMP (cya), transcription initiates equally at S1 and S2; in cAMP-proficient cells (cya+), the S1 initiation increases twofold with a concomitant decrease in S2 initiation. Addition of a saturating amount of exogenous cAMP to cya mutant cells results in a relatively larger switch from S2 to S1. Our results clearly show that while cAMP is an inhibitor of S2, it is not an absolute requirement for transcription initiation at S1, but only acts to increase low-level transcription from the P1 promoter. Using these approaches, we have also studied gal promoter mutants (P211, P18, and P35) which show altered behavior in transcription initiations and in response to cAMP. On the basis of these results, we have discussed models by which transcription initiates at the two overlapping gal promoters (P1 and P2) and discussed how cAMP level modulates the switch between them.