Abstract
The Escherichia coli hns gene encodes the abundant nucleoid-associated DNA-binding protein H-NS. Mutations in hns alter the expression of many genes with unrelated functions and result in a derepression of the proU operon (proVWX) without abolishing the osmotic control of its transcription. We have investigated the interactions of H-NS with the proU regulatory region by deletion analysis of cis-acting sequences, competitive gel retardation assays, and DNase I footprinting. The negative effect of H-NS on proU transcription was mediated by cis-acting sequences within proV but did not depend on the presence of a curved DNA segment upstream of the proU-35 region previously characterized as a target for H-NS binding in vitro. We detected a 46-base pair high affinity H-NS binding region downstream of the proU promoter at the 5' end of the proV gene and a complex array of additional H-NS binding sites which suggest the presence of an extended H-NS nucleoprotein complex. Most of the H-NS binding sites were highly A+T-rich and carried stretches of 5 or more consecutive A-T base pairs. The implications of our results for the osmotic regulation of proU transcription are discussed.
Highlights
Experiments with the protein from E. coli is a highly charged 137-amino acidresidue phage Mu repressor protein suggest that H-NS can act polypeptide (M,= 15,500)that exists insolution predominantly through other, sequence-specific regulatory proteins by either as a homodimer due to strong hydrophobic interactions be- facilitating theirDNA binding or by stabilizing theirproteintween the subunits[6].H-NS binds with high affinity but low DNA interactions [37]
H-NS exhibited a preferential affinity for a 680-bp BglI fragment that carries theproU promoter and extends from position -475 to +202 bp relative to the transcription initiation site (Fig. 1).A small increase(approximately 2-fold) in H-NSconcentration was sufficientto convert the majority of these DNA fragments from the free to the proteinbound form retarded in the gel, which suggests in agreement with previous data (61a) cooperative binding mode of H-NS
Using purified H-NS protein,we have investigated its interactions with thperoU regulatory region by competitive DNA band shift assays anbdy DNase I footprinting analysis
Summary
One of the factorsthat determine thelevel of proU expression is the H-NSprotein. Mutations in thehns structural gene result ina strong increase ofproU transcription, bduotnot abolish osmoregulation ofproU expression [12,22,26].Higgins and co-workers have suggested thatproU transcription is regulated primarily through changes in DthNeA supercoiling of theproU promoter region [11,22,23]. Expression rectly on proU transcription [17]. Ueguchi and Mizuno [40] recently demonstrated that H-NS can selectively inhibit transcription of proU by E. By DNase I protection assays, we identify a n extended H-NS binding region located at the beginning of the proV structural gene which is required for the normal, H-NS-dependent regulation of proU expression. Our data are in agreement witah direct repression model for proU transcription modulation by the H-NS protein
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