We described (1) the properties of a new transcription initiation "factor," factor H, which strongly stimulates RNA synthesis directed by native X-or +80 DNA. Factor H, which was partially purified from a "K succinate eluate" of the DNA-protamine complex that forms during Eacherichia coli RNA polymerase purification, exhibited the following properties: it is a low molecular weight, heat-stable protein, whose simulatory effect is particularly pronounced at low polymerase to DNA ratios. Studies to be described in this paper show that "H" is not a single component but, in fact, represents a family of low molecular weight proteins, two of which-designated as Hi and Hz-appear to be biologically active. Hi and H2 have been characterized and obtained in a high degree of purity.Hi is a neutral and H2 is a basic polypeptide. Both stimulate DNA transcription and readily bind to DNA templates. Several arguments point towards the conclusion that H factors might represent a new class of low molecular weight proteins that facilitate the copying process of polynucleotides by causing appropriate conformational changes, possibly acting as unwinding factors.
MATERIAL AND METHODSChemicals. Triphosphonucleotides were purchased from Sigma.['H]UTP of high specific radioactivity was obtained from the "CEA-Saclay" (France). RNA Polymerase. RNA was prepared from E. coli strain MRE 600 (RNase I-) according to three different procedures. The procedure adapted (1) from Chamberlin and Berg (2) involves use of protamine precipitation, followed by K-succinate elution, ammonium sulfate precipitation, and DEAE-cellulose chromatography. Burgess's technique (3), in which final puri-3643 fication is achieved by two successive glycerol gradient centrifugations, was also used. In some cases, we also used polymerase preparations made according to Babinet (4) and purified through his DEAE-cellulose chromatographic step."H" Factors. Crude H factor was purified from the combined 0.2 and 0.3 M K-succinate eluates of a DNA-protamine precipitate (1). About 50 g of E. coli cells (MRE 600), harvested at the mid-exponential phase, were generally used; 100 ml of 0.2 and 0.3 M K-succinate eluates were pooled, heated for 15 min at 100', and filtered through paper. Factors were precipitated by the addition of solid ammonium sulfate (50 g/100 ml) and adjustment of the pH to 7.5 with diluted KOH. The resulting precipitate was diluted in 0.02 M Tris.HCl (pH 8.0)-0.3 M KC1. The H factors were obtained by Sephadex G-75 filtration (1). Gel filtration at high ionic strength (0.3 M KCl) on a Sephadex G-50 column permitted the resolution of two subfractions, Hi and H2, as illustrated in Fig. 1
Hi protein, a heat-stable low-molecularweight DNA-binding factor previously described by CukierKahn et al. [Proc. Nat. Acad. Sci. USA (1972) In some instances, DNAs from mutants of lac transducing bacteriophages have been used. These mutant strains were Xh8O dtrp X 7713 lac, in which the lactose genes are fused to the tryptophan genes (7); Xh8O dlac p5, a CRP-sensitive lac superpromoter (8, 9), and 080 dlac pr UV6, a CRP-insensitive revertant from the lac-negative LW.CRP, H1 Protein, and RNA Polymerase. Highly purified CRP was a kind gift from B. de Crombrugghe. H1 was obtained as previously described (2). Fig. 1
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