Transcriptional activation by protein-induced DNA bending: evidence for a DNA structural transmission model.

Abstract

Integration host factor (IHF) is a DNAbending protein that binds to an upstream activating sequence (UAS1) and, on a negatively supercoiled DNA Integration host factor (IHF) is a member of a family of DNA-bending proteins that share amino acid similarity with the bacterial HU protein. These are small, basic, minor-groove, nonspecific, DNA-binding proteins that play an architectural role in determining the structure of the bacterial nucleoid (1). IHF is a sequence-specific DNA-binding protein which induces a severe bend in the DNA helix of about 1400 (2). While IHF was initially isolated as a protein of Escherichia coli involved in the higher-order assembly of a nucleoprotein complex required for the integrative recombination of bacteriophage A into the bacterial chromosome, it is now known that IHF is involved in a diverse set of cellular functions, including the regulation of gene expression (1). For example, IHF increases the expression of the ilvGMEDA operon of E. coli, which encodes four of the five enzymes required for the biosynthesis of L-isoleucine and L-valine (3).The ilVPG promoter and the upstream regulatory sequences of the ilvGMEDA operon are shown in Fig. 1 located between base pairs -41 and -59, contains a set of helically phased adenine residues. To disrupt the DNA bend in UAS2, the two adenine residues at positions -44 and -56 and the cytosine residue at -48 were changed to guanine residues by site-directed mutagenesis. To facilitate the reversal of the IHF binding site and replacement of the IHF binding site by the lymphoid enhancer-binding factor (LEF)-1 binding site, the 5'-TTTATT-3' sequence between base pairs -108 and -101 and the 5'-TTGAAA-3' sequence between base pairs -81 and -74 of the wild-type ilv promoter region (4) were changed to Sac I and Xho I endonuclease restriction sites by site-directed mutagenesis. The creation of the Xho I site eliminates the transcriptional activity of a weak, in vitro, promoter (referred to in previous publications as ilvPG1) in UAS1 (3). The loss of this promoter does not affect either the intrinsic DNA bend-mediated or the IHF-induced (Table 1) activation of transcription from the downstream, in vivo, ilvPG promoter [referred to in previous publications as ilvPG2 (3)].sequence centered at base pair -50 that contains two tracts of five and six adenines in helical phase. A DNA bend centered between these phased adenine tracts activates transcription from the ilvPG promoter, in a face-of-the-helix-dependent manner, about 15-fold even in the absence of the upstream IHF site (3). Similar A+T-rich DNA sequences have been observed in the -50 region of several other promoters (5). These other sequences also activate transcription in a face-ofthe-helix-dependent manner about 10-to 30-fold. Ross et al. (5) have suggested that this activation is correlated with the interaction of these curved, upstream, DNA sequences with the a subunit of RNA polymerase, and they have proposed that these "UP" promoter sequences constitute a third promoter recognition ...

DNA-Binding Specificity of Mcm1: Operator Mutations That Alter DNA-Bending and Transcriptional Activities by a MADS Box Protein

“…Alternatively, the decrease in activation obtained with substitutions at this position may be a direct result of the reduced DNA bending. Similar correlations between DNA bending and transcriptional activation have been previously described for integration host factor in prokaryotes and for YY1 in mammalian systems (23,27). …”

Deletion analysis of the fis promoter region in Escherichia coli: antagonistic effects of integration host factor and Fis

“…IHF-mediated activation of ilvp G 2 is not strictly dependent on the helical phasing or distance of the IHF-binding site relative to this promoter, suggesting that interactions between IHF and RNA polymerase or between upstream DNA sequences and RNA polymerase are not required (43). Instead, it appears that an IHF-induced DNA bend alters the structure of the ilvp G 2 Ϫ10 region so as to favor formation of an open complex (44). However, this does not appear to be the mechanism by which IHF stimulates fis P. A 5-bp insertion between fis P and the ihf site caused about a fourfold reduction in transcription in the presence of Fis and about a twofold reduction in its absence.…”

ihfA Gene of the Bacterium Myxococcus xanthus and Its Role in Activation of Carotenoid Genes by Blue Light

“…A direct interaction of IHF with RNA polymerase has been proposed in a few cases in which IHF stimulates transcription without the involvement of other factors (23,26). This is a controversial issue, however, since evidence has been provided for alternative explanations based in IHF-mediated DNA transactions (62,75). The acidic extension of the IhfA of M.…”