The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites.

Abstract

Two sets of experiments have been performed to test the DNA loop model of repression of the araBAD operon of Escherichia coli. First, dimethyl sulfate methylation protection measurements on normally growing cells show that the AraC regulatory protein occupies the aral site in the presence and absence of the inducer arabinose. Similarly, the araO2 site is shown to be occupied by AraC protein in the presence and absence of arabinose; however, its occupancy by AraC is greatly reduced when aral and adjacent sequences are deleted. Thus, AraC protein binds to araO2 cooperatively with some other component of the ara system located at least 60 base pairs away. Second, the mutational analysis presented here shows that the DNA components required for repression of araBAD are aral, araO2, and perhaps the araBAD operon RNA polymerase binding site.The L-arabinose operon of Escherichia coli has long been known to be positively regulated by AraC protein (1). Classical genetic (2-f: and biochemical (5-7) studies have also revealed that th;e) dRAD genes are negatively regulated by AraC and that a site required for this repression is located upstream from all the sites required for induction (2,8,10).Recently, a site required for repression, araO2, was found and was determined to lie 210 base pairs upstream from the AraC binding site required for induction, araI ( Fig. 1) (8). Surprisingly, the behaviors of strains with small deletions and insertions between the araBAD induction region and the araO2 site suggest that repression of the araBAD operon involves the formation of a DNA loop that brings araO2 near the induction region (8). Since repression of a constitutive araBAD promoter requires functional araO2 and araI sites as well as the presence of AraC protein (7) The results of genetic experiments were also as predicted by the DNA loop model. Mutations isolated solely on the basis that they reduced repression were found to lie in the araO2 and araI sites. However, repression-defective mutations also were found in the araBAD operon RNA polymerase binding site, raising the possibility of the involvement of RNA polymerase in repression. Mutations constructed in other known protein binding sites in the regulatory region did not interfere with repression. The results of these standard genetic experiments suggest that only the araI, araO2, and possibly RNA polymerase binding sites are involved in repression. These results strengthen previous results obtained by using "inverse genetics" in which regions of DNA are intentionally mutated and the results observed. MATERIALS AND METHODSMedia, Plasmids, Strains, and General Methods. Media and general methods were as described (11,12). Plasmid pTD3 (9) contains 440 base pairs of the araCBAD regulatory region on a HindIII/EcoRI fragment with PBAD driving galK of the pKO1 vector (13). The plasmid with the araCBAD induction region deleted and used for in vivo footprinting of araO2, pLH1, was made by deleting from to BamHI (position -46) of pTD3. AraO2 deletions of plasmids with araI ...

Two DNA-binding factors recognize specific sequences at silencers, upstream activating sequences, autonomously replicating sequences, and telomeres in Saccharomyces cerevisiae.

“…7D). Also (58). The DNA loop model is also a popular explanation for the orientation and distance independence of enhancers and silencers in eucaryotes (22,63).…”

Suppressor mutations in rpoA suggest that OmpR controls transcription by direct interaction with the alpha subunit of RNA polymerase

“…These differences could be the result of distinct forms or conformations of OmpR or the result of slightly altered placement of OmpR and the polymerase at the various promoters or both. Alternatively, the DNA could act as an allosteric regulator of OmpR that slightly alters the function of the protein, depending on which site OmpR binds (1,24,42).…”

“…Genotype~~~r eference' F-araD139 A(argF-lac)U169 rpsL150 relA flb5301 ptsF25 deoCl MC4100 'I(ompC'-IacZ+)10-25 MC4100 araD+ 4? (ompF'-IacZ+) MC4100 araD+ c1(ompF'-IacZ+) 16-13 rpsE aroE MC4100 araD+ I'(ompF'-lacZ+) 16-13 rpsE MC4100 araD+ 4'(ompF'-lacZ+) 16-13 rpsE zhc-3::TnlO MC4100 zhc-3::TnlO MC4100 4(ompC'-lacZ+)10-25 ompR472 zhc-3::TnJO MC4100 zhc-3::TnlO(XpSG248U) MC4100 ompRiO zhc-3::TnlO (ApSG248U) MC4100 (I(ompC'-IacZ+) [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] envZ473 zhc-3::TnlO MC4100 araD+ F(ompF'-lacZ+) 16 (35). Transformation of plasmid DNA was carried out as previously described (35).…”

ATBF1, a multiple-homeodomain zinc finger protein, selectively down-regulates AT-rich elements of the human alpha-fetoprotein gene.

“…The colinear arrangement of the homeodomains and zinc fingers with the highest sequence homologies between ATBF1 and zfh-2 proteins (17,27) suggests that this may in fact be the case. It is envisaged that the bridging of distal and proximal elements, a step vital for transcriptional regulation (29,39), can be accomplished by a single ATBF1 molecule rather than multiple proteins.…”