The interaction of RNA polymerase II from wheat with supercoiled and linear plasmid templates

Lilley, David M. J.; Houghton, Michael

doi:10.1093/nar/6.2.507

Cited by 25 publications

(8 citation statements)

References 49 publications

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“…DNA torsional strain was created which led to conformational transitions in certain polynucleotide sequences (Luchnik, 1980). Eukaryotic RNA polymerases prefer to transcribe TS DNA (Mandel and Chambon, 1974;Hossenlopp et al, 1974;Lescure et al, 1978;Lilley and Houghton, 1979;Chandler and Gralla, 1981). Thus, the detection of torsionally strained DNA molecules in a transcriptionally active fraction of SV40 minichromosomes is consistent with the above hypothesis.…”

Section: Biological Implicationsmentioning

confidence: 60%

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

Luchnik¹,

Bakayev²,

Zbarsky³

et al. 1982

The EMBO Journal

135

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After treatment of SV40 minichromosomes with DNA topoisomerase I, the superhelicity in the bulk of the DNA extracted from minichromosomes is known to remain unchanged. However, we found that the DNA extracted from a small fraction of SV40 minichromosomes (2‐5%), was almost completely relaxed, and covalently closed as shown by agarose gel electrophoresis. Thus, the DNA in these 2‐5% of SV40 minichromosomes was probably torsionally strained (TS). The proportion of such TS minichromosomes is close to the estimated proportion of transcriptionally active minichromosomes. The distribution of the TS minichromosomes in sucrose gradient coincided with the distribution of transcriptionally active complexes. Both sedimented faster than the majority of minichromosomes. Furthermore, after treatment with topoisomerase I the relaxed minichromosomes could be quantitatively separated from the bulk of material by recentrifugation in a sucrose gradient. A major part of the endogenous RNA polymerase activity was recovered in the relaxed fraction. These data suggest that TS‐minichromosomes correspond to transcriptionally active chromatin. After relaxation with topoisomerase I the TS minichromosomes lacked histones.

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Section: Biological Implicationsmentioning

confidence: 60%

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

Luchnik¹,

Bakayev²,

Zbarsky³

et al. 1982

The EMBO Journal

135

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“…Lilley and Houghton, using wheat germ RNA polymerase II, found at least some residual association (40). Their (46,47) and electron microscopy of binary and ternary complexes of RNA polymerases with DNA (48,49).…”

Section: Discussionmentioning

confidence: 94%

Gel electrophoretic separation of transcription complexes: an assay for RNA polymerase selectivity and a method for promoter mapping

Chelm

Geiduschek

1979

Nucl Acids Res

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ABS ¶[RACI lWe describe a method for analyzing ternary transcription complexes, of RNA polymerase, DNA and nascent RN4-chains, by agarose gel electrophoresis.When the RNA of such complexes is -P-labelled, a simple comparison of the DNA fluorogram with an autoradiogram identifies transcriptionally active DNA molecules and restriction fragments in any mixture. Iwo limitations on the method are described: 1) retardation during electrophoresis of polymerase-DNA complexes relative to their conjugate bare DNA fragments; 2) failure of very large ternary complexes to enter gels.The following potential applications of the method are surveyed: transcription unit (elongation) mapping, separation of RNA molecules in a mixture of transcripts, dinucleotide primer mapping and identification of preferred template conformations. 1IiRODUCI.IONThe transcription reaction catalyzed by DNA-dependent RNA polymerase can be broken down into three steps: initiation, elongation, and termination. In vitro studies with purified hNA polymerases and auxiliary components have been crucial to furthering our understanding of the molecular mechanisms which regulate the selectivity and specificity of this reaction, at least for procaryotic systems (for review see, e.g., 1-3). In this paper we exploit the observation that transcription elongation complexes, that is, ternary complexes, of DNA, RNA polymerase and nascent RNA are stable during a variety of manipulations including electrophoresis through agarose gels. We show how this property can be used to assay transcriptional selectivity and to map transcription units with some advantages in speed and flexibility over already

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“…ED underwinding is an energy requiring process either dependent upon the continuous binding of coiling protein (histone) (25) or linkage reduction by an ATP dependent enzyme (gyrase) (26). It seems likely that the cell should expect some return on its investment of energy in this process, and negative supercoiling has been implicated in replication (27), transcription (28)(29)(30)(31)(32)(33) and recombination (34).…”

Section: Discussion Global Linkage Properties Produce Local Structuramentioning

confidence: 99%

Hairpin-loop formation by inverted repeats in supercoiled DNA is a local and transmissible property

Lilley¹

1981

Nucl Acids Res

130

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Short inverted repeat sequences adopt hairpin stem-loop type structures in supercoiled closed circular DNA molecules, demonstrated by Si nuclease cleavage. Fine mapping of cleavage frequencies is in good agreement with expected cleavage patterns based upon the interaction between an unpaired loop and a sterically bulky enzyme molecule.Whilst the topological properties of underwound DNA circles depend ultimately upon reduced linkage, necessarily a global molecular property, hairpin loop formation is an essentially local property. Thus molecular size is unimportant for the Sl hypersensitivity of the ColEl inverted repeat. Furthermore, a 440 bp Sau3AI, EcoRI fragment of ColEl which contains the inverted repeat has been cloned into pBR322 whereupon it exhibits Sl cleavage similar to ColEl in the supercoiled recombinant molecule. The effect is therefore both local and transmissible. Direct competition between inverted repeats in the recombinant, coupled with close examination of flanking sequences, enables some simple 'rules' for base pairing in hairpin loops to be formulated. Whilst limited G-T and A-C base pairing appears not to be destabilising, A-G, T-C or loop outs are highly destabilising. JLNTRODUCTIONA striking feature of DNA B-form structure is its continuous symmetrical nature (1-3). However from a functional point of view it is equally apparent that discontinuities must be introduced into DNA structure in a sequencedependent manner in order to delineate functionally significant loci such as promoters, replication origins, phasing signals and other putative sites of DNA-protein interaction. Recently it has beccme apparent that DNA may be rather more polymorphic and heterologous (4-6) than has previously been considered. Many fundamental genetic processes such as RNA polymerase initiation events, require localised loss of base pairing, and thus any potential mechanism providing specific opening of the double helix is of considerable importance.I have demonstrated (7) that inverted repeats separated by short nonrepetitious sections of DM are specifically cleaved by the single strand

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The interaction of RNA polymerase II from wheat with supercoiled and linear plasmid templates

Cited by 25 publications

References 49 publications

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: relation to transcriptionally active chromatin.

Gel electrophoretic separation of transcription complexes: an assay for RNA polymerase selectivity and a method for promoter mapping

Hairpin-loop formation by inverted repeats in supercoiled DNA is a local and transmissible property

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