Activation of Escherichia coli leuV transcription by FIS

Abstract

The transcription factor FIS has been implicated in the regulation of several stable RNA promoters, including that for the major tRNALeu species in Escherichia coli, tRNA1Leu. However, no evidence for direct involvement of FIS in tRNA1Leu expression has been reported. We show here that FIS binds to a site upstream of the leuV promoter (centered at -71) and that it directly stimulates leuV transcription in vitro. A mutation in the FIS binding site reduces transcription from a leuV promoter in strains containing FIS but has no effect on transcription in strains lacking FIS, indicating that FIS contributes to leuV expression in vivo. We also find that RNA polymerase forms an unusual heparin-sensitive complex with the leuV promoter, having a downstream protection boundary of approximately -7, and that the first two nucleotides of the transcript, GTP and UTP, are required for formation of a heparin-stable complex that extends downstream of the transcription start site. These studies have implications for the regulation of leuV transcription.

FIG. 1

Sequence of the leuV promoter region. Positions protected by FIS in DNase I footprints and positions of enhanced DNase I cleavage within the FIS site (carets) are indicated. Enhanced DNase I cleavage at −38 and −48 in the presence of RNAP is indicated by vertical arrows. The 2-bp substitution mutant T−71G T−72G reduces FIS binding (Fig. 2). Boundaries of protection by RNAP in the absence (thin underline, −47 to −7) or the presence (thick underline, −47 to +20) of the initiating nucleotides GTP and UTP are indicated. Similarity of the FIS site to a consensus derived from information in references and [Gnn(c/t)(A/g)(a/t)(a/t)(T/A)(t/a)(t/a)(T/c)(g/a)nnC] is indicated by lines between the top and bottom strands. Dots between strands in the FIS site indicate poorly conserved positions in different FIS sites.

FIG. 2

DNase I footprints of FIS bound to wild-type (A) or mutant (B) leuV promoter fragments. BglII-HindIII leuV promoter fragments were obtained from pleuD9 (leuV −109 to +33 [7]) or pHEB3 (leuV −109 to +11, T−71G T−72G [7]) and were ³²P labeled in the bottom (template) strand at the BglII site, approximately 20 bp upstream from leuV position −109. Footprinting reactions were carried out at 22°C, essentially as described previously (35), in a solution of 10 mM Tris-Cl (pH 7.9), 10 mM MgCl₂, 150 mM NaCl, 1 mM dithiothreitol, and 100 μg of bovine serum albumin per ml. FIS was present at the concentrations indicated. Sequence markers were prepared by the method of Maxam and Gilbert (27).

FIG. 3

Effects of FIS on in vitro transcription of wild-type and mutant leuV promoters (wild type, diamonds; T−71G T−72G mutant, triangles; FIS site deletion [−47 endpoint], circles). Transcription was carried out in the absence of FIS or in the presence of the indicated concentrations of FIS by using supercoiled plasmid templates with leuV promoter fragments inserted into the EcoRI and HindIII sites of pRLG770, upstream of the rrnB T1 terminator (36). Plasmids used were pRLG927 (wild-type leuV −109 to +33, obtained from pleuD9 [7]), pRLG930 (leuV −109 to +11, T−71G T−72G, obtained from pHEB3 [7]), and pRLG931 (leuV −47 to +55, obtained from pLC118 [7]). Multiple-round transcription was carried out essentially as described previously (36) except that nucleotide concentrations were 100 μM (for ATP, CTP, and GTP) or 10 μM (for UTP, with [α-³²P]UTP [DuPont, NEN]). Purified FIS was a gift from Reid Johnson (University of California at Los Angeles). leuV transcripts were analyzed on 6.5% acrylamide–7 M urea gels and quantified with a Molecular Dynamics PhosphorImager. The effect of FIS is shown as a ratio of transcript values in the presence and absence of FIS. Results from a representative experiment are shown.

FIG. 4

DNase I footprints of RNAP bound to the wild-type leuV promoter. Complexes were formed with RNAP (10 nM) and the leuV promoter fragment (described in the legend to Fig. 2) in the presence or absence of the initiating nucleotides (500 μM GTP or 500 μM GTP and 50 μM UTP) at 22°C in buffer described in the legend to Fig. 2, except that it contained 30 mM KCl rather than 150 mM NaCl. Where indicated, heparin (10 μg/ml) was added prior to DNase I digestion.