Correlation between the conformation of Escherichia coli −10 hexamer sequences and promoter strength: use of orthophenanthroline cuprous complex as a structural index.

Spassky, Annick; Rimsky, Sylvie; Buc, Henri; Busby, Stephen J. W.

doi:10.1002/j.1460-2075.1988.tb03020.x

Cited by 56 publications

(35 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The region of the "TATA" element was found to be particularly sensitive to the reagent. This is strikingly similar to the -10 sequences of bacterial promoters, which are also hypersensitive to phenanthroline-copper cleavage (48). unique to the adenovirus promoter, as similar cleavage patterns were observed with the human heavy chain immunoglobulin promoter (data not shown).…”

supporting

confidence: 76%

RNA polymerase II-associated proteins are required for a DNA conformation change in the transcription initiation complex.

Buratowski

Sopta

Greenblatt

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Proteins purified on the basis of their affinity for RNA polymerase II effectively substitute for previously defined transcription initiation factors. In two assays, formation of initiation complexes and transcription in vitro, the RNA polymerase II-associated proteins behaved identically to a fraction containing transcription factors HIE and HFE. Both fractions greatly stabilized the association of polymerase with the promoter and were required for the formation of complete initiation complexes. By using the DNA-cleaving reagent phenanthroline-copper in footprinting reactions, the RNA polymerase Il-associated proteins were shown to be required for a DNA conformation change near the initiation site of the promoter. Based on similarity to the prokaryotic transcription complex, this conformation change is likely to represent a transition from a closed to an open complex.The mechanism of transcription initiation by RNA polymerase II is one of the most fundamental processes in the eukaryotic cell, yet one of the least well understood. In addition to polymerase, several accessory initiation factors have been identified by in vitro reconstitution assays (1-9). However, separated and purified factors in sufficient quantities for extensive biochemical analysis have been difficult to obtain. The recent cloning of the genes for several of the initiation factors has improved this situation (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).Despite the limitations imposed by the use of partially purified factors, significant progress has been made in defining some of the events leading to transcription initiation in vitro. The first step is recognition of the "TATA" element by transcription factor TFIID (2,7,(21)(22)(23)(24)(25)(26)(27)(28). TFIIA also appears to exert its stimulatory effect at this step, presumably through its interaction with TFIID (2,7,8,22,(29)(30)(31)(32). A stable complex of the promoter, TFIID, and TFIIB can be observed by native gel electrophoresis, in the presence and absence of TFIIA (refs. 18, 22, and 32 and S.B., unpublished results). RNA polymerase bound to this complex can also be resolved in native gels, as can complexes formed by the subsequent binding of TFIIE/F. DNase I protection assays of the initiation complexes indicate that TFIIE/F binds downstream of the polymerase molecule, between positions +20 and +30 relative to the initiation site (22).Once the initiation complex has assembled, an ATPdependent "activation" step occurs (33-35). Activation coincides with a loss of protein-DNA interactions between positions +20 and +30 of the promoter (22,26,36,37) and probably consists of an ATP-dependent dissociation of TFIIE or TFIIF (22). Initiation can occur once complexes are activated.At some point in the initiation process, the DNA duplex must be unpaired to allow base pairing of the elongating transcript with the template strand. In prokaryotic transcription initiation, this unwinding is well characterized and known as the "closed-to-open" complex transition (38). This kinetically import...

show abstract

supporting

confidence: 76%

RNA polymerase II-associated proteins are required for a DNA conformation change in the transcription initiation complex.

Buratowski

Sopta

Greenblatt

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Additional 5-phenyl-OP-Cu hypersensitive sites are also found around the transcription start site for each metal-activated protein when RNAP is added to the reaction. This cleavage by Cu-OP at positions Ϫ3 to Ϫ7 on the template strand and ϩ4 to ϩ5 on both strands correlates well with the presence of an open, transcriptionally active complex at other promoters (56,57).…”

Section: Discussionsupporting

confidence: 56%

DNA Distortion Mechanism for Transcriptional Activation by ZntR, a Zn(II)-responsive MerR Homologue in Escherichia coli

Outten¹,

Outten²,

O’Halloran³

1999

Journal of Biological Chemistry

182

197

View full text Add to dashboard Cite

MerR-like DNA distortion mechanisms have been proposed for a variety of stress-responsive transcription factors. The Escherichia coli ZntR protein, a homologue of MerR, has recently been shown to mediate Zn(II)-responsive regulation of zntA, a gene involved in Zn(II) detoxification. To determine whether the MerR DNA distortion mechanism is conserved among MerR family members, we have purified ZntR to homogeneity and shown that it is a zinc receptor that is necessary and sufficient to stimulate Zn-responsive transcription at the zntA promoter. Biochemical, DNA footprinting, and in vitro transcription assays indicate that apo-ZntR binds in the atypical 20-base pair spacer region of the promoter and distorts the DNA in a manner that is similar to apo-MerR. The addition of Zn(II) to ZntR converts it to a transcriptional activator protein that introduces changes in the DNA conformation. These changes apparently make the promoter a better substrate for RNA polymerase. We propose that this zinc-sensing homologue of MerR restructures the target promoter in a manner similar to that of other stress-responsive transcription factors. The ZntR metalloregulatory protein is a direct Zn(II) sensor that catalyzes transcriptional activation of a zinc efflux gene, thus preventing intracellular Zn(II) from exceeding an optimal but as yet unknown concentration.

show abstract

“…To examine the location of ParB on the DNA in the I ϩ B1 and I ϩ B2 complexes, we used OP-Cu to footprint complexes directly in the electrophoresis gel. OP-Cu is a chemical nuclease that interacts with DNA through the minor groove, where it cleaves the DNA backbone (26,27). Access of OP-Cu to the DNA can be blocked sterically by proteins or by an alteration in the geometry of the DNA that narrows the minor groove.…”

Section: B1/hb1mentioning

confidence: 99%

Stoichiometry of P1 Plasmid Partition Complexes

Bouet

Surtees

Funnell

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

The P1 plasmid prophage is faithfully partitioned by a high affinity nucleoprotein complex assembled at the centromere-like parS site. This partition complex is composed of P1 ParB and Escherichia coli integration host factor (IHF), bound specifically to parS. We have investigated the assembly of ParB at parS and its stoichiometry of binding. Measured by gel mobility shift assays, ParB and IHF bind tightly to parS and form a specific complex, called I ؉ B1. We observed that as ParB concentration was increased, a second, larger complex (I ؉ B2) formed, followed by the formation of larger complexes, indicating that additional ParB molecules joined the initial complex. Shift Western blotting experiments indicated that the I ؉ B2 complex contained twice as much ParB as the I ؉ B1 complex. Using mixtures of ParB and a larger polyhistidine-tagged version of ParB (His-ParB) in DNA binding assays, we determined that the initial I ؉ B1 complex contains one dimer of ParB. Therefore, one dimer of ParB binds to its recognition sequences that span an IHF-directed bend in parS. Once this complex forms, a second dimer can join the complex, but this assembly requires much higher ParB concentrations.

show abstract

Correlation between the conformation of Escherichia coli −10 hexamer sequences and promoter strength: use of orthophenanthroline cuprous complex as a structural index.

Cited by 56 publications

References 41 publications

RNA polymerase II-associated proteins are required for a DNA conformation change in the transcription initiation complex.

RNA polymerase II-associated proteins are required for a DNA conformation change in the transcription initiation complex.

DNA Distortion Mechanism for Transcriptional Activation by ZntR, a Zn(II)-responsive MerR Homologue in Escherichia coli

Stoichiometry of P1 Plasmid Partition Complexes

Contact Info

Product

Resources

About