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

Chelm, Barry K.; Geiduschek, E. Peter

doi:10.1093/nar/7.7.1851

Cited by 44 publications

(19 citation statements)

References 39 publications

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“…Detection of electrophoretic bands (TIMING 30 min to several days, depending on the radioactivity of the sample) 10. At the end of electrophoresis, remove the gel and plate assembly from the electrophoresis device and dry it thoroughly with paper towels.…”

Section: Electrophoresis (Timing 30 Min-5h Depending On the Electrophmentioning

confidence: 99%

“…1). The current, widely-used assay differs little from that originally described by Fried and Crothers 7 and Garner and Revzin 8 , although precursors to the technique can be found in the earlier literature [9][10][11] . Mobility-shift assays are often used for qualitative purposes, although under appropriate conditions they can provide quantitative data for the determination of binding stoichiometries, affinities and kinetics 3,6,12 .…”

Section: Introductionmentioning

confidence: 99%

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Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

2007

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The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32 P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.

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Section: Electrophoresis (Timing 30 Min-5h Depending On the Electrophmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

2007

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“…To show that altering the DnaA box 1 weakened or abolished binding to the enhancer, we applied the agarose gel retardation assay to DnaA (Fig. 2) Chelm and Geiduschek (3) and has been subsequently used in a number of laboratories, including our own, in the analysis of complexes of DNA with IHF (9) and IT (14). Figure 2 shows that DnaA forms one major complex with the wt enhancer carrying one DnaA box but no detectable complexes with the mutant R6K enhancer, which carries no sequences with a match to the DnaA consensus of greater than six of nine.…”

Section: Schaefer and Messer (40) (T/c)(t/c)(a/t/c)t(a/c)c(a/g) (A/cmentioning

confidence: 99%

Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein

Levchenko

Filutowicz

1994

J Bacteriol

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Replication of the -y origin ofEscherichia coli plasmid R6K requires i protein, encoded by the R6Kpir gene, and many host factors, including DnaA protein. zr has dual roles, activating replication at low levels and inhibiting replication at high levels. The inhibitory function of w is counteracted by integration host factor and a specific sequence of the origin called the enhancer. This 106-bp DNA segment contains a binding site for DnaA protein (DnaA box 1). In this study, we mutated this site to determine if it was required for the enhancer's function. Using -y origin derivative plasmids with the DnaA box 1 altered or deleted, we show that this site is necessary to protect the origin against levels of wild-type wF protein that would otherwise inhibit replication. To show that the base substitutions in DnaA box 1 weakened the binding of DnaA, we developed a new application of the agarose gel retardation assay. This quick and easy assay has broad applicability, as shown in binding studies with DNA fragments carrying a different segment of the R6K origin, the chromosomal origin (oriC), or the pUC origin. The gel retardation assay suggests a stoichiometry of DnaA binding different from that deduced from other assays.Many replication origins have a bipartite organization, including an essential segment and an auxiliary segment which augments replication. Such enhancers have been discovered in organisms and systems as diverse as, for example, the bacteriophage fl (8, 23), the Staphylococcus aureus plasmid pT181 (21), and the yeast autonomously replicating sequence ARS121 (49). In each of these cases, the enhancer carries, or is believed to carry, a protein-binding site (20, 37a, 49, 50).The Escherichia coli plasmid R6K contains three origins of replication, a, y, and ,3 ( Fig. 1), which all require the initiator protein 7r, encoded by the R6K pir gene. All three origins require the essential 277-bp core segment, which contains repeated binding sites (iterons) for TT (14,19,33); each origin also needs a unique cis-acting DNA segment in addition to the core (28,29,36,41,42,44). When the flanking ot and P sequences are removed, the remaining y origin can replicate autonomously (18,43,46) if thepir gene is provided in cis or in trans (22,29). The y origin responds to the intracellular concentration of uz protein, and it contains the most critical information required for replication (12,28). Therefore, we use the y origin system (for the most recent review, see reference 10) as the simplest model derived from R6K to study the mechanisms regulating initiation of replication of an iteron-containing origin.The -y origin consists of two modules, the essential 277-bp core segment and the adjoining 106-bp enhancer (51a) (Fig. 1). The enhancer is not required for the a or ,B origin (36, 41, 42, 44). The isolated core is able to replicate if the level of wild-type (wt) wT is decreased (Sla), a situation which reduces the stringency of replication control (12). At the normal level of wt 7r (4,000 to 10,000 IT (9,11,25), the p...

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“…Ternary complexes remain intact during gel electrophoresis (11,13,50). When void volume fractions were subjected to agarose gel electrophoresis, most of the radioactivity comigrated with either plasmid or chromosomal DNA (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…(c) Fractions (25 ,ul of each) 8 through 19 were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (10% acrylamide) (39) and stained by the silver staining procedure of Morrissey (38). Proteins which appeared to elute in the same fractions as the DNA are marked by a dot between lanes 11 tial loss of gpS5-dependent ternary complexes during these manipulations. Thus, this hybridization analysis principally established that the correlation of transcriptional activity with the state of supercoiling was generally shared by gp55-dependent and other transcription of the plasmid DNA.…”

Section: Legend)mentioning

confidence: 99%

Bacteriophage T4 late transcription from plasmid templates is enhanced by negative supercoiling

1988

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Concurrent viral replication is normally required to activate bacteriophage T4 late promoters; replication is thought to provide a template structure which is competent for late transcription. Transcription from plasmid-borne T4 late promoters, however, is independent of replication in vivo and in vitro. In this work, we have shown that, when the late gene 23 promoter is located on a plasmid, its utilization in vivo depends upon the ability of host DNA gyrase to maintain some degree of negative superhelicity. This suggests that an alternative pathway exists for activation of late promoters: DNA which is under sufficient negative torsional stress is already competent for late transcription. We also describe a method for isolating ternary complexes of plasmid DNA, RNA polymerase, and nascent RNA which have initiated transcription in vivo. The topoisomer distribution of such ternary complexes prepared from T4-infected cells showed that, late in infection, transcriptional activity resides primarily in the subset of the plasmid population with the most negatively supercoiled topoisomers. However, the overall transcriptional pattern in these ternary complexes indicated that both vector and T4 sequences are actively transcribed. Much of this transcriptional activity could be independent of gp55, the T4-specific RNA polymerase-binding protein that confers late promoter recognition.During infection of Escherichia coli by bacteriophage T4, a program of modifications to the host RNA polymerase generates three major temporal classes of phage transcripts (reviewed in references 7, 15, and 42). Late transcription requires the products of T4 genes 33, 45, and 55. Gene product 55 (gp55) confers specificity for late T4 promoters on RNA polymerase core (26). The roles of gp33 and gp45 in transcription remain unclear, but gp45 is known also to play a role in DNA replication as an accessory protein to the replication complex (1). Late promoters share the sequence TATAAATA centered at about position -10 from the start of transcription, but require no position -35 sequence (12,14).Late transcription is also distinguished by a requirement for concurrent viral DNA replication (45). Late transcription can be uncoupled from replication in genetic backgrounds which are thought to stabilize nicks in the DNA. These observations led to the hypothesis that late transcription requires a competent template which has been activated by DNA replication (46). Uncoupling can also be achieved by placing the promoter on a plasmid template (21, 23). Late transcription from plasmid templates in vivo is otherwise faithful in its initiation sites and in regulation by genes 33, 45, and 55 (14, 23). Additionally, supercoiled plasmids are efficient templates for in vitro transcription with purified T4 late RNA polymerase (24). Therefore it appears that plasmid templates are intrinsically competent for late transcription.The topoisomer profile of plasmids changes drastically after T4 infection. We have argued that there are two reasons for this topoisomerizat...

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

Cited by 44 publications

References 39 publications

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions

Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein

Bacteriophage T4 late transcription from plasmid templates is enhanced by negative supercoiling

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