IHF-binding sites inhibit DNA loop formation and transcription initiation.
Transcriptional activation of enhancer and sigma(54)-dependent promoters requires efficient interactions between enhancer-binding proteins (EBP) and promoter bound sigma(54)-RNA polymerase (E sigma(54)) achieved by DNA looping, which is usually facilitated by the integration host factor (IHF). Since the lengths of the intervening region supporting DNA-loop formation are similar among IHF-dependent and IHF-independent promoters, the precise reason(s) why IHF is selectively important for the frequency of transcription initiation remain unclear. Here, using kinetic cyclization and in vitro transcription assays we show that, in the absence of IHF protein, the DNA fragments containing an IHF-binding site have much less looping-formation ability than those that lack an IHF-binding site. Furthermore, when an IHF consensus-binding site was introduced into the intervening region between promoter and enhancer of the target DNA fragments, loop formation and DNA-loop-dependent transcriptional activation are significantly reduced in a position-independent manner. DNA-looping-independent transcriptional activation was unaffected. The binding of IHF to its consensus site in the target promoters clearly restored efficient DNA looping formation and looping-dependent transcriptional activation. Our data provide evidence that one function for the IHF protein is to release a communication block set by intrinsic properties of the IHF DNA-binding site.
Huo
Yi-Xin
YX
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, PR China.
Zhang
Yuan-Tao
YT
Xiao
Yan
Y
Zhang
Xiaodong
X
Buck
Martin
M
Kolb
Annie
A
Wang
Yi-Ping
YP
D001483:Base Sequence; D001665:Binding Sites; D003500:Cyclization; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D034982:Integration Host Factors / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004269:DNA, Bacterial; D034982:Integration Host Factors