Volume 1, Issue 1, Pages 99-107 (December 1997) Transcription Activation or Repression by Phage Φ29 Protein p4 Depends on the Strength of the RNA Polymerase–Promoter Interactions  María Monsalve, Belén Calles, Mario Mencía, Margarita Salas, Fernando Rojo  Molecular Cell  Volume 1, Issue 1, Pages 99-107 (December 1997) DOI: 10.1016/S1097-2765(00)80011-8

Figure 1 Schematic Represententation of the Promoters Analyzed (A) Characteristics of the wild-type late A3 and early A2c promoters. (B) Derivatives of the A3 promoter; in promoter A3Δ10 the binding site for protein p4 was moved 10 bp closer to the transcription initiation site (from position −82 to −72), introducing at the same time a XhoI restriction site; in promoter A3−35+ a −35 consensus sequence for σA-RNAP was introduced. (C) Derivatives of the A2c promoter. Promoter A2c-p4A3 was obtained by eliminating the binding site for protein p4 and substituting it with that of the A3 promoter, positioning it at −81, one helix turn upstream as compared with P A2c. In promoter A2c−35−, the consensus sequence for σA-RNAP was eliminated and substituted with an unrelated sequence. Molecular Cell 1997 1, 99-107DOI: (10.1016/S1097-2765(00)80011-8)

Figure 2 Activity of the Promoter Mutants Derived from P A3 and P A2c and Effect of Protein p4 or of Its Mutant Derivative R120Q In vitro run-off assays were carried out with the linear DNA templates (10 nM) indicated in the Experimental Procedures and σA-RNAP (35 nM); protein p4 or its mutant derivative R120Q were added where indicated at 0.8 μM. The run-off transcript corresponding to each promoter is shown. Molecular Cell 1997 1, 99-107DOI: (10.1016/S1097-2765(00)80011-8)

Figure 3 Effect of Protein p4 on the Activity of the Promoter Mutants Derived from P A3 and P A2c In vitro run-off assays were carried out with the linear DNA templates (10 nM) indicated in the Experimental Procedures and σA-RNAP (70 nM); protein p4 was added at the concentrations indicated in each panel. Activation is indicated as the amount of transcripts observed in the presence of protein p4 relative to those obtained in its absence (X-fold), while repression is shown as % activity relative to that obtained in the absence of protein p4. Molecular Cell 1997 1, 99-107DOI: (10.1016/S1097-2765(00)80011-8)

Figure 4 Role of the RNAP α-CTD on the p4-Dependent Activation or Repression of the Promoters Derived from P A3 and P A2c Transcription reactions were performed as described in Figure 2 except that the DNA template was present at 3.3 nM and reconstituted RNAPs were used. The RNAP harboring a wild-type α subunit is indicated as α-wt, while that reconstituted with a mutant α subunit lacking the last 15 residues from the C-end is indicated as α-Δ15. Reconstituted RNAPs were added in amounts giving transcription levels over p4-independent promoters similar to those observed with a wild-type non-reconstituted RNAP (seeMencí a et al. 1996a; Monsalve et al. 1996b). The transcript generated from each promoter is shown. Molecular Cell 1997 1, 99-107DOI: (10.1016/S1097-2765(00)80011-8)

Figure 5 DNaseI Footprinting of the Complexes Formed by Protein p4 and/or σA-RNAP at Promoters Derived from P A3 and P A2c An end-labeled DNA fragment (1 nM) containing the indicated promoter was incubated for 10 min at 37°C in the presence of the proteins denoted prior to addition of DNAse I. Protein p4 was present at 1.3 μM and σA-RNAP at 70 nM. The results obtained for promoters A3, A3−35+, and A3Δ10 are shown in (A), while promoters A2c, A2c−35−, or A2c-p4A3 are shown in (B). The binding sites of protein p4 and RNAP are indicated, as well as some of the positions that become hypersensitive to DNase I upon binding of protein p4 and/or RNAP. The discontinuous line indicates a region where the footprints for protein p4 and RNAP overlap. Molecular Cell 1997 1, 99-107DOI: (10.1016/S1097-2765(00)80011-8)

Figure 5 DNaseI Footprinting of the Complexes Formed by Protein p4 and/or σA-RNAP at Promoters Derived from P A3 and P A2c An end-labeled DNA fragment (1 nM) containing the indicated promoter was incubated for 10 min at 37°C in the presence of the proteins denoted prior to addition of DNAse I. Protein p4 was present at 1.3 μM and σA-RNAP at 70 nM. The results obtained for promoters A3, A3−35+, and A3Δ10 are shown in (A), while promoters A2c, A2c−35−, or A2c-p4A3 are shown in (B). The binding sites of protein p4 and RNAP are indicated, as well as some of the positions that become hypersensitive to DNase I upon binding of protein p4 and/or RNAP. The discontinuous line indicates a region where the footprints for protein p4 and RNAP overlap. Molecular Cell 1997 1, 99-107DOI: (10.1016/S1097-2765(00)80011-8)

Figure 6 Effect of Protein p4 on the Abortive Transcripts Produced at the A3−35+ Promoter A DNA fragment containing the A3−35+ promoter (10 nM) was incubated for 10 min at 37°C in the presence of RNAP (70 nM) and, where indicated, protein p4 (1.3 μM). Nucleotides were added (200 μM each except for [α-32P]UTP, which was added at 2, 4, or 8 μM), and after 30 min at 37°C the reaction was stopped and the products loaded onto a denaturing gradient (6%-23%) polyacrylamide gel. The position of some of the abortive products and of the full-length run-off transcript are indicated. Molecular Cell 1997 1, 99-107DOI: (10.1016/S1097-2765(00)80011-8)

Figure 7 Protein p4 Inhibits Promoter Clearance at the A2c-p4A3 Promoter The complexes formed by protein p4 and RNAP at promoter A2c-p4A3 in the presence of the four NTPs (200 μM each) were analyzed by DNase I footprinting; those formed at the A2c promoter are shown as controls. End-labeled DNA fragments (1 nM) were incubated for 10 min at 37°C in the absence or presence of protein p4 (1.3 μM) and/or RNAP (9 nM), the four NTPs were added, and after a further 5 min incubation the complexes formed were analyzed by DNase I footprinting. The regions protected from DNase I attack by protein p4 and RNAP are indicated, as well as some of the positions that become hypersensitive to DNase I upon binding of protein p4 or RNAP to the promoter. The discontinuous line indicates a region where the footprints for protein p4 and RNAP overlap. Molecular Cell 1997 1, 99-107DOI: (10.1016/S1097-2765(00)80011-8)

Figure 8 Effect of Protein p4 on RNAP Clearance at the A3−35+ Promoter An end-labeled DNA fragment containing the A3−35+ promoter (1 nM) was incubated for 10 min at 37°C in the absence or presence of protein p4 (1.3 μM) and RNAP (0.35, 0.7, 1.4, 3.5, or 7 nM). The four NTPs (200 μM) were added where indicated, and after 5 additional min at the same temperature promoter occupancy was monitored by DNase I footprinting. The binding regions of protein p4 and RNAP, as well as some of the positions that become hypersensitive to DNase I upon protein binding, are indicated. Molecular Cell 1997 1, 99-107DOI: (10.1016/S1097-2765(00)80011-8)