Reaching Out

Eustance, Rebecca J.; Bustos, Silvia A.; Schleif, Robert

doi:10.1006/jmbi.1994.1584

Cited by 23 publications

(15 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Members of the family for which in vitro or in vivo footprinting assays are available (AraC, RhaR, RhaS, MelR, SoxS, and Ada) have at least two features in common: they function in vivo as a dimer, and the stretch of nucleotides covered by a monomer of the regulatory protein at the regulated promoter is between 15 and 20 bp long. However, within this set of bases, short motifs seem to confer critical base recognition for DNA-protein interactions (15)(16)(17)(18)(19)(20)(21). This seems also to be the case for Pm/XylS interactions.…”

Section: Discussionmentioning

confidence: 99%

Critical Nucleotides in the Upstream Region of the XylS-dependent TOL meta-Cleavage Pathway Operon Promoter as Deduced from Analysis of Mutants

González-Pérez

Ramos

Gallegos

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

The Pm promoter, dependent on TOL plasmid XylS regulator, which is activated by benzoate effectors, drives transcription of the meta-cleavage pathway for the metabolism of alkylbenzoates. This promoter is unique in that in vivo transcription is mediated by RNApolymerase with different sigma factors. In vivo footprinting analysis shows that XylS interacted with nucleotides in the ؊40 to ؊70 region. In vivo and in vitro methylation of Pm shows extensive methylation of T at position ؊42 in the bottom strand, suggesting that it represents a key distortion point that may favor XylS/ RNA polymerase interactions. Methylation of T ؊42 was highest in cells bearing XylS and in the presence of an effector. Gs in the ؊47 to ؊61 region appeared to be more protected in cells harboring XylS in the presence than in the absence of the effector. Almost 100 mutants in the Pm region between ؊41 and ؊78 were generated; transcriptional analysis of these mutants defined the XylS target as two direct repeats with the sequence TGCAN 6 GGNCA. These motifs cover the ؊70 to ؊56 and the ؊49 to ؊35 regions. Single point mutations revealed that nucleotides located at ؊49 to ؊46 and at ؊59, ؊60, ؊62, and ؊70 are the most critical for appropriate XylS-Pm interactions.

show abstract

Section: Discussionmentioning

confidence: 99%

Critical Nucleotides in the Upstream Region of the XylS-dependent TOL meta-Cleavage Pathway Operon Promoter as Deduced from Analysis of Mutants

González-Pérez

Ramos

Gallegos

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Samples were heated for 2 min at 90°C prior to separation of the products by electrophoresis on a 6% polyacrylamide gel containing 8 M urea. 32 P end-labeled HpaIIdigested pUC DNA (Geneworks) was used as a marker to estimate the size of the RNA transcripts. Gels were dried and exposed overnight to a phosphorimager screen.…”

Section: Bacteria-hms174 (De3) Plyssmentioning

confidence: 99%

Establishment of Lysogeny in Bacteriophage 186

Shearwin

Egan²

2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

The CII protein of bacteriophage 186 is a transcriptional activator of the helix-turn helix family required for establishment of the lysogenic state. DNA binding by 186 CII is unusual in that the invertedly repeated half sites are separated by 20 base pairs, or two turns of the DNA helix, rather than the one turn usually associated with this class of proteins. Here, we investigate quantitatively the DNA binding properties of CII and its interaction with RNA polymerase at the establishment promoter, p E . The stoichiometry of CII binding was determined by sedimentation equilibrium experiments using a fluorescein-labeled oligonucleotide and purified CII. These experiments indicate that the CII species bound to DNA is a dimer, with additional weak binding of a tetrameric species at high concentrations. Examination of the thermodynamic linkages between CII selfassociation and DNA binding shows that CII binds to the DNA as a preformed dimer (binding free energy, 9.9 kcal/mol at 4°C) rather than by association of monomers on the DNA. CII binding induces in the DNA a bend of 41 (؎ 5) degrees. The spacing between the binding half sites was shown to be important for CII binding, insertion or removal of just 1 base pair significantly reducing the affinity for CII. Removal of 5 or 10 base pairs between binding half sites eliminated binding, as did insertion of an additional 10 base pairs. CII binding at p E was improved marginally by the presence of RNA polymerase (⌬⌬G ‫؍‬ -0.5 (؎ 0.3) kcal/mol). In contrast, the binding of RNA polymerase at p E was undetectable in the absence of CII but was improved markedly by the presence of CII. Thus, CII appears to recruit RNA polymerase to the promoter. The nature of the base pair changes in mutant phage, selected by their inability to establish lysogeny, are consistent with this mechanism of CII action.Understanding the mechanisms involved in the control of gene expression requires knowledge of the physicochemical interactions occurring between the components. Even relatively simple prokaryotic systems usually involve several linked protein-protein and protein-DNA interactions, the mechanistic and thermodynamic features of which must be defined in order to gain a full appreciation of the system (see, for example, Refs. 1-6). We were interested in understanding the nature of the interactions involved in the self-association and DNA binding of bacteriophage 186 CII protein, the concentration of which inside the bacterial host cell ultimately decides the fate of that infected cell.Upon infection of the host cell, coliphage 186 can pursue one of two independent but interchangeable life cycles: lytic development or lysogeny. The region of the 186 genome controlling the fate of the phage (and its host) consists of two face-to-face promoters, p R and p L (Fig. 1), which control transcription of the lytic and lysogenic operons, respectively. As p R is a much (approximately 200-fold) stronger promoter than p L (8), the choice between lysis and lysogeny does not reflect simple competition bet...

show abstract

“…Interestingly, proteins that are expected to be more flexible than wild type Lac repressor such as AraC 109,[119][120][121] and Lac repressor mutants 122 present a different shape in their gene expression curves and, consequently, in their looping energies. In both cases the looping energy does not display a minimum.…”

mentioning

confidence: 99%

Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity

et al. 2006

View full text Add to dashboard Cite

The mechanical properties of DNA play a critical role in many biological functions. For example, DNA packing in viruses involves confining the viral genome in a volume (the viral capsid) with dimensions that are comparable to the DNA persistence length. Similarly, eukaryotic DNA is packed in DNA-protein complexes (nucleosomes) in which DNA is tightly bent around protein spools. DNA is also tightly bent by many proteins that regulate transcription, resulting in a variation in gene expression that is amenable to quantitative analysis. In these cases, DNA loops are formed with lengths that are comparable to or smaller than the DNA persistence length. The aim of this review is to describe the physical forces associated with tightly bent DNA in all of these settings and to explore the biological consequences of such bending, as increasingly accessible by single-molecule techniques.

show abstract

Reaching Out

Cited by 23 publications

References 0 publications

Critical Nucleotides in the Upstream Region of the XylS-dependent TOL meta-Cleavage Pathway Operon Promoter as Deduced from Analysis of Mutants

Critical Nucleotides in the Upstream Region of the XylS-dependent TOL meta-Cleavage Pathway Operon Promoter as Deduced from Analysis of Mutants

Establishment of Lysogeny in Bacteriophage 186

Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity

Contact Info

Product

Resources

About