The Selectivity of Transcription

Chamberlin, Michael J.

doi:10.1146/annurev.bi.43.070174.003445

Cited by 529 publications

(229 citation statements)

References 176 publications

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“…RNA polymerase-DNA open complexes are very unstable at low temperature and in the presence of high ionic strengths while complexes that have initiated transcripts are not (1,19). These properties were used to examine whether open complexes, defined as described in the experiment in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Initiation of mRNA synthesis involves a series of reactions between RNA polymerase holoenzyme (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) and DNA sequences termed "promoters." The steps in this interaction are thought to include (1): (a) The formation of a relatively loose but specific "closed complex" between the polymerase and the promoter. (b) Conversion The costs of publication of this article were defrayed in part by the payment of page charges.…”

mentioning

confidence: 99%

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RNA polymerase binding sites in λp lac 5 DNA

Jones¹,

Chan²,

Rothstein³

et al. 1977

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

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The in vitro binding of the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) to fragments of Aplc5 DNA generated by restriction endonucleases HindII and HindIII has been studied by a filter binding technique. The results are consistent with RNA polymerase binding at p-, the INT promoter (pI), several sites in the b2 region, the mis promoter, the oop promoter (or po), and pro. Binding was also observed on some fragments that are not known to contain active promoters, including the fragment from the CIII-tL region. Some of these binding reactions might also be explained by interaction of RNA polymerase with termination sites. Additional polymerase binding sites have been detected by examining which HindII and HindIII sites were not cleaved when digestion was performed after RNA polymerase had been bound to the DNA. This technique revealed polymerase binding at PL, at PR9 at a site between R and cos, and at a site at the junction of the y and cIII-tL fragments. A comparison of the location of polymerase binding fragments with the partial denaturation map of the X genome indicates that RNA polymerase binding sites are located within A-T rich regions. It is suggested that RNA polymerase binding is a function both of specific sequences (where recognition occurs) and of the base composition of the surrounding regions (which affects the stability of the helix at the specific site). Initiation of mRNA synthesis involves a series of reactions between RNA polymerase holoenzyme (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) and DNA sequences termed "promoters." The steps in this interaction are thought to include (1) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. fined segments by the restriction enzymes HindII and HindIII. RNA polymerase was incubated with the fragments, heparin was added, and the mixture was passed through a nitrocellulose filter, on which the "open complexes" were retained. The DNA fragments associated with RNA polymerase in these complexes were identified by polyacrylamide gel electrophoresis. These complexes were also tested for their initiation potential. Similar types of analyses have been performed previously (2-4).The second technique used is called a "protection experiment" and is identical to one used by Allet and Solem (5) and Allet et al. (6). Some promoters are known to contain HindII sites (6-12) and therefore would not be detected by an RNA polymerase binding experiment. These sites can be discerned by adding RNA polymerase prior to the addition of the HindII and HindIII enzymes and examining the final digest for HindII sites that have not been cleaved. It is assumed that this protection represents specific open complex formation although no competitor has been added and the initiation potential has not been tested.Having identified RNA polymerase...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

RNA polymerase binding sites in λp lac 5 DNA

Jones¹,

Chan²,

Rothstein³

et al. 1977

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

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“…Such questions provoked the suggestion that nuclear DNA might be supercoiled [61] and lie behind the continuing search for torsionally strained or 'dynamic' chromatin. Early studics showed conclusively that supercoiling stimulates transcription in uitro [62,631 ; whether it does in vivo, and whether some chromatin is torsionally strained, are now the issues.…”

Section: Loops and Supercoils In Native Chromatinmentioning

confidence: 99%

The nucleoskeleton and the topology of transcription

Cook

1989

European Journal of Biochemistry

108

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Transcription is conventionally believed to occur by passage of a mobile polymerase along a fixed template. Evidence for this model is derived almost entirely from material prepared using hypotonic salt concentrations. Studies on subnuclear structures isolated using hypertonic conditions, and more recently using conditions closer to the physiological, suggest an alternative. Transcription occurs as the template moves past a polymerase attached to a nucleoskeleton; this skeleton is the active site of transcription. Evidence for the two models is summarised. Much of it is consistent with the polymerase being attached and not freely diffusible. Some consequences of such a model are discussed.The accepted model for transcription contains three essential participants -the template, polymerase and nascent RNA [I, 21. Transcription is initiated by a diffusible polymerase binding to a promoter; the polymerase then processes along the DNA (Fig. 1 A). As knowledge has increased, other occasional participants have been added (e. g. transcription factors, topoisomerases) but not any other central players. Evidence for this model comes almost entirely from studies using hypotonic salt concentrations, largely because chromatin aggregates in physiological concentrations and so becomes difficult to use. In contrast, studies on preparations isolated using hyper-or iso-tonic salt concentrations suggest that active RNA polymerase is associated with a nucleoskeleton and is not freely diffusible. This nucleoskeleton is seen as the active site of RNA synthesis and transcription occurs by movement of DNA past the attachcd polymerase (Fig. 1 B).Ultimately models will be distinguished by reconstructing efficient transcription in vitro from purified constituents. Proof of the model involving an attached polymerase will require measurement ofits association constant for a skeleton. In the meantime, the two models can be distinguished operationally by determining whether the polymerase is freely diffusible or attached to a larger structure. Much evidence for the 'text-book' model is consistent with the alternative; this alternative has a number of important consequences and these are examined. Discussion centrcs on RNA polymerase 11, the enzyme that transcribes most eukaryotic genes, but applies equally to other polymerases. ARTEFACTSStructure within isolated nuclei cannot be discussed sensibly without some consideration of problems caused by artefacts. They arise because nuclei and chromatin aggregate in physiological salt concentrations 131 ; therefore unphysiological conditions are almost invariably used. ControversyCorrespondence to P. R. Cook, Sir William Dunn School of Pathology, Universlty of Oxford, South Parks Road, Oxford OX1 3RE. UK centres on whether structures seen In vitro are any more than isolation artefacts. RNA, DNA and protein, each concentrated in the nucleus at about 0.1 g/ml, might be expected to aggregate as soon as ion concentrations are altered. (For reviews of the controversy, see [4-61.) Nuclei are usually isol...

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“…The best characterized protein is rho which has a monomer molecular weight of 50000 as compared with 70000 for this inhibitor, and which binds to phosphocellulose [4] which this inhibitor does not. Rho factor can act to terminate synthesis at defined sites on DNA molecules such as T7 [24] and lambda [4] as well as sites which are polar in vivo [25], but does not terminate on poly[d-(A-T)] [3]. In contrast this inhibitor does not recognize any sites located within the early T7 transcript and in addition works on both single-stranded DNA and poly[d(A-T)].…”

Section: Discussionmentioning

confidence: 99%

Isolation and Properties of an Inhibitor of Escherichia coli RNA Polymerasè

Crimaldi

Ihler

1976

European Journal of Biochemistry

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An inhibitor of the Escherichia coli RNA polymerase has been isolated from E. coli and has been partially characterized. The inhibitor, a polypeptide of molecular weight 70000, acts to shut off RNA synthesis at about the time that the first round of RNA synthesis is over, preventing any further RNA synthesis. The inhibitor apparently does not recognize specific termination sequences in the DNA template, since it works equally well with double-stranded DNA, single-stranded DNA and poly[d(A-T)] as templates for RNA synthesis, and because the RNA molecules synthesized from T7 DNA appear to be terminated at the same site either in the presence or absence of the inhibitor. Several experiments indirectly indicate that the inhibitor may reversibly bind to the RNA polymerase at the termination step, in a ratio of approximately one inhibitor molecule per polymerase molecule.The core RNA polymerase of Escherichia coli consist of three different polypeptides, p' (molecular weight 165000), p (molecular weight 155000) and M (molecular weight 39 000) with the subunit composition pP'a2 [1,2]. Core polymerase is the minimum form of the enzyme capable of polymerization of ribonucleoside triphosphates into RNA, but a variety of other polypeptides are known regulate or modify the specificity and activity of core enzyme, Among these are polypeptides which play a role in initiation, such as sigma and the adenosine 3' : 5'-monophosphate acceptor protein and others which play a role in termination, such as rho, as well as various relatively uncharacterized stimulatory and inhibitory proteins. (For a recent review of RNA synthesis, see [3].) Rho factor [4] is required for efficient termination of RNA synthesis at specific sites on the DNA and helps regulate the amounts and kinds of RNA species synthesized. We have isolated a new polypeptide which has some features in common with rho, but which does not appear to recognize specific sites on the DNA. It may play a role in regulating the amount of active RNA polymerase. Enzyme. DNA-dependent RNA polymerase (EC 2.7.7.6). MATERIALS AND METHODS Purification10 g casamino acids, 6 g NazHP04 . 7 H20, 3.2 g KH2P04, 10 ml glycerol and 0.3 mg MgS04. Cell growth was stopped at a cell density of 6 x lo8 cells/ ml by the addition of ice. The cells were harvested using a DeLaval cream separator and frozen at -20 "C. 50-60 g of frozen cells, were mixed with 100 g of hydrochloric-acid-washed glass beads (3M Co. Superbrite 100) and 50 ml of grinding buffer (0.05 M Tris pH 7.9,0.25 M KC1,O. 1 M dithiothreitol, 0.1 mM EDTA and 5 % glycerol) and were homogenized in a solid COz/acetone/water-cooled Waring blender for 5 min at low speed and 10 min at high speed. DNase I (0.5 mg) was added and the supernatant decanted after 30 min at 4 "C. The glass beads were reextracted with the addition of 25 ml of grinding buffer. Debris and ribosomes were removed by centrifugation for 2 h at 30000 rev./min in a Spinco 30 rotor and the supernatant was fractionated by the addition of solid (N&)zS04. The material which preci...

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The Selectivity of Transcription

Cited by 529 publications

References 176 publications

RNA polymerase binding sites in λp lac 5 DNA

RNA polymerase binding sites in λp lac 5 DNA

The nucleoskeleton and the topology of transcription

Isolation and Properties of an Inhibitor of Escherichia coli RNA Polymerasè

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