NORMAL GENOMIC

• Niu W, Kim Y, Tau G, Heyduk T, Ebright RH
• Transcription activation at class II CAP-dependent promoters: twointeractions between CAP and RNA polymerase.
• Cell. 1996; 87: 1123-34
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• At Class II catabolite activator protein (CAP)-dependent promoters, CAPactivates transcription from a DNA site overlapping the DNA site for RNApolymerase. We show that transcription activation at Class IICAP-dependent promoters requires not only the previously characterizedinteraction between an activating region of CAP and the RNA polymerasealpha subunit C-terminal domain, but also an interaction between a second,promoter-class-specific activating region of CAP and the RNA polymerasealpha subunit N-terminal domain. We further show that the two interactionsaffect different steps in transcription initiation. Transcriptionactivation at Class II CAP-dependent promoters provides a paradigm forunderstanding how an activator can make multiple interactions with thetranscription machinery, each interaction being responsible for a specificmechanistic consequence.

• Zhou Y, Merkel TJ, Ebright RH
• Characterization of the activating region of Escherichia coli catabolitegene activator protein (CAP). II. Role at Class I and class IICAP-dependent promoters.
• J Mol Biol. 1994; 243: 603-10
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• CAP-dependent promoters can be divided into classes based on the positionof the DNA site for CAP. In class I CAP-dependent promoters, the DNA sitefor CAP is located upstream of the DNA site for polymerase; the DNA sitefor CAP can be located at various distances from the transcription startpoint, provided that the DNS site for CAP and the DNA site for RNApolymerase are on the same face of the DNA helix. In class IICAP-dependent promoters, the DNA site for CAP overlaps the DNA site forRNA polymerase, replacing the -35 determinants for binding of RNApolymerase. In previous work, we have shown that a surface loop consistingof amino acid residues 152 to 166 of CAP is essential for transcriptionactivation at the best-characterized class I CAP-dependent promoter, thelac promoter, and we proposed that this surface loop makes directprotein-protein contact with RNA polymerase in the ternary complex of lacpromoter, CAP, and RNA polymerase. Here, we show that the surface loopconsisting of amino acid residues 152 to 166 is essential fortranscription activation at other class I CAP-dependent promoters and at aclass II CAP-dependent promoter. We show further that the effects ofalanine substitutions of residues 152 to 166 are qualitatively identicalat the lac promoter and other class I CAP-dependent promoters, but aredifferent at a class II CAP-dependent promoter. We propose that thesurface loop consisting of residues 152 to 166 makes identical molecularinteractions in transcription activation at all class I CAP-dependentpromoters, irrespective of distance between the DNA site for CAP and thetranscription start point, but makes a different set of molecularinteractions in transcription activation at class II CAP-dependentpromoters.

• Ushida C, Aiba H
• Helical phase dependent action of CRP: effect of the distance between theCRP site and the -35 region on promoter activity.
• Nucleic Acids Res. 1990; 18: 6325-30
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• A plasmid carrying a CRP-dependent promoter fused to the lac structuralgenes was manipulated to construct a set of spacing mutants that havevarying lengths between the CRP binding site and the -35 region. Thelengths of the spacer were changed over 45 bp by inserting or deletingnucleotides. DNase I footprinting analysis revealed that the spacer lengthdid not affect the binding of cAMP-CRP to the CRP site. The effect of thespacer length on transcription activation by cAMP-CRP was tested in vivoby beta-galactosidase and quantitative S1 assays with crp+ and delta crpcells harboring plasmids. Insertions or deletions of non-integral helicalturns, which displace the CRP site onto the opposite face of DNA helixcompared to the original promoter, eliminated completely the activation oftranscription. In contrast, changing the spacer length by integral helicalturns allowed the promoter to respond to CRP, although the degree ofactivation varied with the length of the spacer. We conclude thatstereospecific positioning of CRP and RNA polymerase on the DNA helix isstrictly required for CRP action. The data support a model that CRPstimulates transcription by directly contacting RNA polymerase.

• Contreras A, Drummond M
• The effect on the function of the transcriptional activator NtrC fromKlebsiella pneumoniae of mutations in the DNA-recognition helix.
• Nucleic Acids Res. 1988; 16: 4025-39
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• We have constructed mutations in what we predict to be the DNA-recognitionhelix of Klebsiella pneumoniae NtrC, which regulates transcription frompromoters under global nitrogen control. Mutations which disrupt the helixlead to complete loss of function. All point mutants tested were able toactivate transcription from the sigma 54-dependent glnA promoter, but onlythose retaining some ability to recognise NtrC binding sites, as evidencedby their ability to repress the ntrB promoter and the upstream glnApromoter, were able to activate the nifL promoter. One mutant, whichcontained an amino acid substitution in the turn of the DNA-binding motifas well as in the recognition helix, suppressed mutations in the NtrCbinding sites upstream from the nifL promoter, but only if both sites boreequivalent transitions. This confirms that the DNA-binding motif for thisclass of transcriptional activator has been correctly identified andsuggests that binding of NtrC can be cooperative.

• Hope IA, Struhl K
• GCN4, a eukaryotic transcriptional activator protein, binds as a dimer totarget DNA.
• EMBO J. 1987; 6: 2781-4
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• The eukaryotic transcriptional activator protein, GCN4, synthesized invitro from the cloned gene, binds specifically to the promoters of yeastamino acid biosynthetic genes. Previous analysis of truncated GCN4derivatives localized the DNA binding domain to the C-terminal 60 aminoacids and revealed that the size of the GCN4 derivative and theelectrophoretic mobility of the protein-DNA complex were inverselyrelated. This observation was utilized here to develop a novel method fordetermining the subunit structure of DNA binding proteins. A mixture ofwild-type GCN4 protein and a smaller GCN4 derivative generated threecomplexes with DNA, two corresponding to those observed when the proteinsare present individually and one new complex of intermediate mobility.This extra complex results from the heterodimer of the two GCN4 proteinsof different sizes, demonstrating that GCN4 binds DNA as a dimer. Thecontacts sufficient for dimerization were localized to the 60 C-terminalamino acid, DNA binding domain, suggesting that dimerization of GCN4 is acritical aspect of specific DNA binding. Furthermore, stable GCN4 dimerswere formed in the absence of target DNA. These observations suggest astructural model of GCN4 protein in which a dimer binds to overlapping andnon-identical half-sites, explaining why GCN4 recognition sites actbidirectionally in stimulating transcription.

• Travers A, Klug A
• Nucleoprotein complexes. DNA wrapping and writhing.
• Nature. 1987; 327: 280-1

• Joachimiak A, Kelley RL, Gunsalus RP, Yanofsky C, Sigler PB
• Purification and characterization of trp aporepressor.
• Proc Natl Acad Sci U S A. 1983; 80: 668-72
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• We have isolated homogeneous trp aporepressor from an overproducing strainof Escherichia coli carrying a plasmid containing trpR preceded by tandemtrp operon promoters. Dye-affinity and ion-exchange chromatography wereused in conjunction with a gel electrophoresis assay in which therepressor, when bound to the trp operator, protects an Rsa I restrictionsite from endonuclease cleavage. Crystals suitable for x-ray diffractionstudies were grown from a variety of concentrated salt solutions.Hydrodynamic properties and electrophoretic analysis of unmodified andcovalently crosslinked aporepressor show that the free aporepressor has anisoelectric point of 5.9 and is a dimer containing two identical12.5-kilodalton subunits in the presence or absence of L-tryptophan. Therepressor . operator complex binds poorly to nitrocellulose filters, butrestriction-site protection studies indicate that, in the presence oftryptophan, one dimer is bound to the operator site with an apparentdissociation constant less than 2 X 10(-9) M. Preliminary equilibriumdialysis experiments suggest that tryptophan binds to the aporepressorwith a dissociation constant of 1.6 X 10(-5) M.

• Eilen E, Krakow JS
• Effects of cyclic nucleotides on the conformational states of the alphacore of the cyclic AMP receptor protein.
• Biochim Biophys Acta. 1977; 493: 115-21
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• The alpha core gragment produced by limited proteolysis contains thecyclic AMP binding domain and the two buried sulfhydryl groups of thecyclic AMP receptor protein. The buried sulfhydryl groups of the alphacore react with 5,5'-dithio-bis(2-nitrobenzoic acid) after denaturation by3 M urea or digestion with subtilisin. The rate of sulfhydryl modificationin the presence of 3 M urea or subtilisin is markedly decreased in thepresence of cyclic nucleotides which are proposed to tighten theconformation of the alpha core. Incubation of the alpha core in 3 M ureaor dithionitrobenzoic acid does not affect cyclic AMP binding whiledithionitrobenzoic acid plus 3 M urea inhibits cyclic AMP bindingsuggesting a role for the buried sulfhydryls in cyclic AMP binding ortheir proximity to the cyclic AMP binding domain of the alpha core. Thedata are consistent with a ligand-induced conformational change in thealpha region of the native cyclic AMP receptor protein that is requiredfor DNA binding.