Transcription activation by phage phi29 protein p4 is mediated by interaction with the alpha subunit of Bacillus subtilis RNA polymerase.

Abstract

We have found that protein p4 can induce the binding of purified B. subtilis a subunit to the late A3 promoter and that this depends on an interaction held between the protein p4 carboxyl-end and the C-terminal region of the a subunit. The use of reconstituted RNAPs with nested deletions at the a subunit carboxyl-end has indicated that the last 15 residues of RNAP a subunit are required for protein p4-dependent transcription activation. The implications of these results for the transcription activation mechanism of protein p4 are discussed. METHODSCloning and Construction of Deletion Mutants ofB. subtilis rpoA Gene, Coding for RNAP a Subunit. Plasmid pT7Ba was obtained by assembling into the pT7-7 expression vector (26) two DNA fragments from plasmids pSA136 and pSW102 (provided by Chester Price, University of California), containing the 5'-and 3'-segments of B. subtilis rpoA gene (27) 6616The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Functional interactions between a phage histone-like protein and a transcriptional factor in regulation of phi 29 early-late transcriptional switch

“…As expected, when RNAP was the only protein incubated with the DNA fragment, binding of RNAP to PA2b and PA2c was detected (Fig. 3, lane 2; Rojo and Salas 1995;Monsalve et al 1996a). When p4 (330 nM) and RNAP were simultaneously present, the set of hypersensitive bands that characterize binding of p4 immediately upstream of RNAP at PA2c and PA3 was observed; in addition, the −82 hypersensitive band at PA2b was lost as a conse- Figure 3.…”

“…Thus, the presence of RNAP is not essential for p4 to exert its effect on the binding of p6 to this DNA region. Because p4 binds with very low affinity to its site at PA2c and with high affinity to its site at PA3 in the absence of RNAP Monsalve et al 1996a), these observations suggest that it is the binding of p4 to the latter site that is important in facilitating binding of p6.…”

“…Arrows at right indicate DNase I hypersensitivities created on binding of protein p6 (numbered relative to the transcription start site at PA2c) and binding of protein p4 to PA3 (numbered relative to the transcription start sites at PA2c/PA3). Numbers in boxes are the specific bands that were densitometrically scanned to obtain data presented in Table 1. quence of p4 binding at PA3 Monsalve et al 1996a). Because the presence of p4 is required for stabilization of RNAP at PA3, protection downstream of the p4-binding site at this promoter was now observed because of the binding of RNAP to PA3.…”

“…It should be noted that although the footprint assay presented here was performed in the absence of nucleotides (to visualize binding of RNAP to PA3, which would otherwise escape the promoter), the same results regarding PA2c were obtained in the presence of nucleotides. Basically, the footprint that characterizes protein p4 holding the RNAP at PA2c (Monsalve et al 1996a) was replaced by that of protein p6 bound to this promoter (data not shown).…”

“…8). When p6 is absent, binding of p4 to its PA2c site causes repression by preventing RNAP promoter clearance (Monsalve et al 1996a). However, in the presence of both p4 and p6, it is p6 that directly represses PA2c by blocking access to RNAP.…”