Characterization of ribonucleic acid polymerase-T7 promoter binary complexes

Cech, Carol L.; McClure, William R.

doi:10.1021/bi00552a023

Cited by 72 publications

(42 citation statements)

References 14 publications

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“…The biphasic dissociation behavior has also been observed in the telomere-binding protein purified from O. nova (Raghuraman and Cech 1989}. Protein-DNA complexes have a wide range of half-lives: <1 sec for T4 gene 32 protein-DNA complexes (Lohman 1984); a few minutes for the lactose repressor-operator DNA complex (Whitson and Matthews 1986); and on the order of 10 hr for RNA polymerase-T7 promoter complexes (Cech and McClure 1980). The 100-hr half-life observed for the telomeric complex is very long in this context.…”

Section: Cooperative Binding Of A-and [3-subunitsmentioning

confidence: 74%

Oxytricha telomere-binding protein: separable DNA-binding and dimerization domains of the alpha-subunit.

Fang

Gray²,

Cech³

1993

Genes Dev.

108

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A telomere-binding protein heterodimer of 56-kD (,v) and 41-kD (~) subunits binds to the single-stranded (T4G4)2 terminus of each Oxytricha nova macronuclear DNA molecule. The a-subunit by itself binds to telomeric DNA. The 13-subunit alone does not bind to DNA specifically but interacts with the ot-subunit to form a very stable ternary complex. We show that the formation of e~-~-DNA ternary complex is extremely cooperative. Furthermore, the binary complex (~-DNA) has a dissociation half-life of much less than 1 min; addition of the f~-subunit increases the half-life to -100 hrs. Libraries of plasmids with random deletions of the open reading frame for the ~-subunit were introduced into Escherichia coli, and extracts were subsequently checked for both protein expression and DNA-binding activity with or without added ~-subunit. The ~-subunit was found to contain two structurally separable domains with distinct functions. The amino-terminal two-thirds is necessary and sufficient for sequence-specific DNA binding. The carboxy-terminal one-third is responsible for a/~-subunit interactions. When expressed separately in E. coil, purified, and mixed together, these two domains reconstitute the activity of the wild-type c~-subunit (trans-complementation in vitro). The amino-terminal two-thirds of the f~-subunit is necessary and sufficient both for a/13-subunit interactions and for ternary complex formation. We conclude that the ~-subunit of the telomere-binding protein, like many transcription factors, has separable DNA-binding and protein-protein interaction domains.[Key Words: Oxytricha; telomere-binding protein; DNA-binding domain; protein-protein interaction domain; domain complementation in trans]

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Section: Cooperative Binding Of A-and [3-subunitsmentioning

confidence: 74%

Oxytricha telomere-binding protein: separable DNA-binding and dimerization domains of the alpha-subunit.

Fang

Gray²,

Cech³

1993

Genes Dev.

108

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“…The use of both poly[d(A-T)] and heparin in determining the lifetimes was necessitated by our reluctance to rely solely on heparin challenge data because there is evidence that, for some promoters, heparin directly attacks the open or closed complexes or both (11,16). We conclude that the x3 and PR open complex dissociation rates were unaffected by heparin at 50 ,gg/ml.…”

Section: Resultsmentioning

confidence: 99%

In vitro comparison of initiation properties of bacteriophage lambda wild-type PR and x3 mutant promoters.

Hawley¹,

McClure²

1980

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

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Promoters are regions of DNA to which RNA polymerase binds to begin RNA synthesis. The structural features of promoters and the details of the polymerase-DNA interaction are subjects of great interest because the initiation of transcription is an important control point in the regulation of gene expression. Promoters recognized by Escherichia coli RNA polymerase contain two regions of sequence homology: (i) the Pribnow box, a six-base-pair sequence centered at -10 with respect to the startpoint of transcription, and (ii) another six-base-pair sequence located around -35. RNA polymerase interacts with bases in the Pribnow box and -35 region, as determined by chemical probe experiments. The importance of these sequences to in ivo polymerase recognition is demonstrated by the occurrence of most promoter mutations within these two regions (1, 2).The frequency of transcription initiation at some promoters is regulated by accessory proteins, such as repressors and activators. Other promoters appear not to require additional protein factors; indeed, mutations that affect transcription from a promoter both in vio and in vitro demonstrate that the DNA sequence alone can directly determine the frequency of initiation on a promoter. One such mutant, x3, is a base-pair change in the -35 region of PR, the major rightward promoter of bacteriophage X (3). We studied the initiation properties of x3 and PR to determine what steps in the RNA polymerase-promoter interaction have been affected by this base pair change.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. These data were analyzed in a way that allowed the equilibrium constant for the initial binding (KI = k1/k_1) to be quantitated separately from the rate of open complex formation (k2). This latter process is referred to as an isomerization because we do not know whether we have measured the rate of DNA melting or another rate-determining conformational change. The results of our study indicate that the main effect of the x3 mutation in vitro is a decrease in K, to 5% of the wild-type value and a decrease in k2 to 20%. MATERIALS AND METHODS DNA Templates and Enzyme. The source of X PR was the 890-base-pair DNA fragment isolated after Hae III digestion of purified bacteriophage X DNA. The X x3 promoter was obtained by Hae III digestion of a pBR313-derived plasmid, pRB70, constructed by Roger Brent (Harvard University). This plasmid contained an insertion of XcI857x3 DNA spanning the region from the EcoRI site in the 0 gene to the Bam I site to the left of the N gene. The x3 mutation abolishes HindII cutting within the promoter (3); this characteristic was confirmed for the A Hae III 890 fragment obtained from this plasmid (unpublished data). DNA fragments resulting from Hae III digestion of phage or plasmid DNA were separated on a 3.5% polyacrylamide gel and extracted from the gel with phenol (6). The DNA was separated from UV-absorbing impurities...

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“…Thus, using heparin challenge experiments (as described in Ref. 27), we have measured the stability of the FNR-dependent open complexes at FF(Ϫ41.5) by determining the backward rate constant for isomerization (k Ϫ2 ). To do this, open complexes were allowed to form in the presence of either FNR* or FNR* T118A/S187A.…”

mentioning

confidence: 99%

Role of Activating Region 1 of Escherichia coliFNR Protein in Transcription Activation at Class II Promoters

Wing

Green

Guest

et al. 2000

Journal of Biological Chemistry

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FNR is an Escherichia coli transcription factor that activates gene expression in response to anaerobiosis at a large number of promoters by making direct contacts with RNA polymerase. At class II FNR-dependent promoters, where the DNA site for FNR overlaps the ؊35 element, activating region 1 of FNR is proposed to interact with the C-terminal domain of the RNA polymerase ␣-subunit. Using a model class II FNR-dependent promoter, FF(؊41.5), we have performed in vivo and in vitro experiments to investigate the role of this interaction. Our results show that FNR, carrying substitutions in activating region 1, is compromised in its ability to promote open complex formation and thus to activate transcription. Abortive initiation assays were used to assess the contribution of activating region 1 of FNR to open complex formation. A new method for the purification of the FNR protein is also described.Many bacterial gene regulatory proteins activate transcription by making direct contact with RNA polymerase holoenzyme (RNAP).1 A large number of these activators contact RNAP via the C-terminal domain of the ␣-subunit (␣CTD) (1). One of the best studied examples of this type of activator is the Escherichia coli cAMP receptor protein, CRP (reviewed in Refs. 2 and 3). In response to glucose starvation, CRP is triggered by cAMP and activates transcription initiation at more than 100 different promoters. In its active form, CRP binds as a dimer to specific DNA sequences found at target promoters; the consensus site is a 22-base pair sequence that is organized as an 11-base pair inverted repeat. At CRP-dependent promoters, CRP activates transcription by making several direct contacts with RNAP. One of these contacts involves the interaction of a single surface-exposed ␤-turn of CRP (residues 156 -164) with ␣CTD. This region in CRP is known as activating region 1 (AR1). The role of AR1 of CRP is to enhance open complex formation at target promoters simply by increasing the initial binding of RNAP (reviewed in Ref. 2).The FNR protein is another global activator of gene expression in E. coli, which regulates transcription initiation in response to oxygen starvation (4, 5). FNR belongs to the same family of transcription factors as CRP, and the two proteins have related amino acid sequences (6). Although the high resolution structure of the FNR protein has not been determined, several crystallographic structures for CRP have been solved (7-9) and sequence alignments suggest that FNR and CRP have similar three-dimensional structures (4, 6). Like CRP, FNR regulates transcription as a dimer and recognizes a 22-base pair binding sequence at target promoters. However, dimerization of FNR is triggered by the anaerobic acquisition of a 2ϩ center in each FNR subunit (10 -12). By analogy to CRP, during transcription initiation at FNR-dependent promoters it is proposed that FNR interacts with the ␣CTD of RNAP. Genetic analysis suggests that the amino acid side chains involved in this interaction are located in three adjacent surface-exposed...

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Characterization of ribonucleic acid polymerase-T7 promoter binary complexes

Cited by 72 publications

References 14 publications

Oxytricha telomere-binding protein: separable DNA-binding and dimerization domains of the alpha-subunit.

Oxytricha telomere-binding protein: separable DNA-binding and dimerization domains of the alpha-subunit.

In vitro comparison of initiation properties of bacteriophage lambda wild-type PR and x3 mutant promoters.

Role of Activating Region 1 of Escherichia coliFNR Protein in Transcription Activation at Class II Promoters

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