Transcriptional Interference between Convergent Promoters Caused by Elongation over the Promoter  Benjamin P Callen, Keith E Shearwin, J.Barry Egan  Molecular Cell  Volume 14, Issue 5, Pages 647-656 (June 2004) DOI: 10.1016/j.molcel.2004.05.010

Figure 1 Convergent Promoters from the Developmental Switches of Bacteriophage 186 and P2 A. DNA sequence of 186 convergent promoters pR and pL and details of the fragment used (Dodd et al., 1990). Bent arrows indicate the start sites of transcription, predicted −10 and −35 hexamers are boxed and the promoter mutations used are indicated. The sequences of the NheI promoter fragments used to clone combinations of 186 and P2 promoters in Figure 4 are also shown. B. DNA sequence of P2 convergent promoters pe and pc and details of the fragment used. Start sites (+1) of pe and pc transcription were determined by primer extension (A. A. Berg, personal communication) Molecular Cell 2004 14, 647-656DOI: (10.1016/j.molcel.2004.05.010)

Figure 2 Promoter Activities of Constructs Testing pR Interference of pL Transcription Experiments test A. wild-type promoter activities, B. increased spacing, C. divergent transcription, D. divergent antisense transcription, E. clipped overlapping convergent transcription, F. insertion of tA− DNA, and G. insertion of tA+ DNA. The transcriptional activities of the indicated constructs were measured by LacZ assay of strains containing the appropriate λRS45ΔYA-pBC2-lacZ prophage. λRS45ΔYA-pBC2RIII-lacZ prophages containing a RNaseIII cleavage site (indicated as scissors on RNA) between the promoter inserts and lacZ were used in experiment E and for all rightward activities. The RNaseIII cleavage site is reported to reduce potential context effects from different constructs (Linn and St.Pierre, 1990) and was used such that the effect of tA+ on rightward activity could be more accurately determined. Leftward values indicate the LacZ activities of constructs with pL expressing LacZ in the presence of active pR (pR+) or inactive pR (pR−). Rightward values are the LacZ activities of constructs with pR expressing LacZ (some values not determined, n.d.). For each experiment the fold interference was calculated as the ratio of pR− and pR+ leftward activities, as described in Experimental Procedures. In the construct diagrams, numbers above the line indicate the position from the +1 site of pR and numbers below the line indicate the position from the +1 of pL. Wavy lines indicate transcripts from pR or pL. Regions of pL/pR transcript complementarity are indicated by black wavy lines. Relevant BamHI (B) and SmaI (S) restriction sites are indicated. tA+ indicates an active trpA terminator and tA− is the mutated trpA terminator. Molecular Cell 2004 14, 647-656DOI: (10.1016/j.molcel.2004.05.010)

Figure 3 In Vitro Transcription of pR, pL and pc A. Diagram of some of the templates used for in vitro transcription. pBC1 is a cloning vector we constructed to facilitate the provision of supercoiled templates for this study. For the representatives shown, pBC1.pRpL carried an insert of the phage 186 DNA fragment bearing wild-type pR and pL in the native arrangement (Figure 1A) and pBC1.pc pe-, an insert bearing wild-type pc and mutated pe in their native arrangement (Figure 1B). Expected sizes of the relevant transcripts produced from each plasmid are shown. B. In vitro transcript pattern obtained for templates pBC1 only, pBC1.pRpL, pBC1.pRpL−, pBC1.pR−pL, pBC1.pcpe− and pBC1.pc−pe− (lanes 1 to 6, respectively). In vitro transcription assays were performed in 10 μl volumes for each template as described in Experimental Procedures. RNAP and DNA was pre-incubated at 37°C for 30 min to allow maximum open complex formation at all relevant promoters and elongation was terminated after 60 min to allow complete firing and elongation from all relevant promoters. C. Rate of formation of active heparin resistant complexes at pL(pR–) (triangles), pRNA1 (diamonds) and pc(pe−) (circles). 90 μl in vitro transcription reactions containing template DNA (pBC1.pR−pL or pBC1.pcpe−) were incubated at 37°C and initiated by the addition of RNAP at time 0. Aliquots of the reaction were taken at various times, added to the NTP/heparin mix (final volume 10 μl) and allowed to elongate for 60 min. The amount of relevant full-length transcripts accumulated was quantitated, and expressed as a percentage of the maximum for that transcript and then plotted against pre-incubation time. D. Rate of full length transcript production from heparin-resistant complexes formed at pR(pL-)(squares), pL(pR−)(triangles) and pc(pe-) (circles). 60 μl in vitro transcription reactions containing RNAP and DNA (pBC1.pRpL-, pBC1.pR−pL or pBC1.pe−pc) were pre-incubated at 37°C for 30 min to allow complete open complex formation for all relevant promoters. Upon the addition of NTP/heparin mix, 7 μl aliquots were added to stop/load buffer at times indicated on the graph and the transcription pattern was analyzed. Relevant full-length transcripts were quantitated and plotted against elongation time. Data from two independent experiments was fitted to a first order exponential rise to a maximum using SigmaPlot v4 and from this relationship the time for 50% transcript production (t50%) was calculated. E. Rate of full length transcript production from heparin-resistant complexes formed at pR(pL−) (squares), pR(pL+) (circles), pL(pR+) (diamonds), and pL(pR−) (triangles). Details are as for Figure 3D but using templates pBC1.pRpL−, pBC1.pRpL or pBC1.pR−pL. F. In vivo potassium permanganate footprinting of open complexes at pR and pL was performed on pBC2.pLpR- and pBC2.pLpR+ templates using a primer which anneals upstream of pL. The lanes marked A, C, G and T are dideoxy sequencing reactions performed on pBC2 pLpR+ using the same primer. A second primer, annealing within the bla gene, was included in the primer extension reactions to generate the reference band. Molecular Cell 2004 14, 647-656DOI: (10.1016/j.molcel.2004.05.010)

Figure 4 Comparison of Interference between P2 and 186 Promoters Promoter activities measured by LacZ assay showing interference in the native P2 promoters pepc (A) and in constructs containing combinations of pe, pc, pR and pL convergent promoters, with a centrally located NheI linker between convergent promoter fragments (B-E). Details are as for Figure 2. Molecular Cell 2004 14, 647-656DOI: (10.1016/j.molcel.2004.05.010)