Figure 4.

Lack of functional ‘memory’ during enhancer-dependent activation of the glnAp2 promoter. (A) Time-courses of single-round and multiple-round transcription of supercoiled pLR100 plasmid having 110 bp enhancer–promoter spacing. The experimental strategy for comparison of the rates of single- and multiple-round transcription is outlined at the top. Reaction mixtures were incubated in the presence of all nucleotides (multiple-round) or with ACG mixture only (single-round). Use of an ACG mixture instead of ATP prevents conversion of RP_o back to RP_c and thus allows comparison of single- and multiple-round transcription under similar conditions. The reaction was terminated by adding heparin with subsequent incubation for 5 min. Heparin prevents formation of new initiation complexes but allows completion of already initiated transcripts. Labeled transcripts were analyzed in a denaturing PAGE. The loading control (a 227 bp end-labeled DNA fragment) was added to the reaction mixtures immediately after terminating the reaction. (B) Initiation of transcription occurs at similar rates during the first and subsequent rounds of transcription on supercoiled DNA. Quantitative analysis of the data is shown in (A). The intensities of the bands containing 484 nt transcripts were analyzed using a PhosphorImager.