<i>In vitro</i>transcription close to the melting point of DNA: analysis of<i>Thermotoga maritima</i>RNA polymerase—promoter complexes at 75°C using chemical probes

Meier, Thomas; Schickor, Peter; Wedel, Andrew; Cellai, Luciano; Heumann, Hermann

doi:10.1093/nar/23.6.988

Cited by 24 publications

(27 citation statements)

References 25 publications

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“…5). Melting by both sigma 70 and sigma 54 holoenzymes begins near Ϫ11 and spreads downstream (7)(8)(9)(10), suggesting that sigma binding to the optimal Ϫ12͞Ϫ11 fork junction probably starts the opening process. The adjacent nontemplate strand (in the case of sigma 70) then should be bound, perhaps only when conditions allow the inhibitory Ϫ10 position gate to be unlocked.…”

Section: Discussionmentioning

confidence: 99%

“…In this process, holoenzyme first binds to the promoter to form a closed complex and then opens a segment roughly from position Ϫ11 to ϩ3. Chemical modifications reveal structural distortions in closed complexes near position Ϫ11, suggesting that nucleation of promoter opening occurs near the upstream boundary of the open region (7)(8)(9)(10). The activity responsible for this critical step is not known.…”

mentioning

confidence: 99%

“…The activity responsible for this critical step is not known. Opening then may extend unidirectionally to expose the transcriptional start site (7,8). The sequences on the nontemplate strand of the Ϫ10 consensus element, which extends from Ϫ12 to Ϫ7, are known to have an important influence (11).…”

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confidence: 99%

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Promoter opening via a DNA fork junction binding activity

Guo

Gralla

1998

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

153

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The rate-limiting step in transcriptional initiation typically is opening the promoter DNA to expose the template strand. Opening is tightly regulated, but how it occurs is not known. These experiments identify an activity, recognition of specific DNA fork junctions, and suggest that it is critical to bacterial promoter opening. This activity is both sequence and structure specific; it recognizes the bases that constitute the upstream double-stranded͞ single-stranded boundary of the open complex. Promoter mutations known to reduce opening rates lead to comparable reductions in fork junction binding affinity. The activity acts to establish the upstream boundary of melted DNA and works in conjunction with two single-stranded DNA binding activities that recognize separately the two melted strands. The junction binding activity is contained within the sigma factor component of the holoenzyme. The activity occurs in both a typical prokaryotic transcription system and in a eukaryotic-like bacterial system that responds to enhancers and needs ATP. Thus DNA opening catalyzed by fork junction binding may occur in a variety of systems in which DNA must be opened to be copied.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Promoter opening via a DNA fork junction binding activity

Guo

Gralla

1998

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

153

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“…This indicates that conformational changes in the DNA that accompany initiation of transcription such as promoter melting are determined by the polymerase rather than the DNA sequence (Meier et al, 1995).…”

Section: Number Of Promoter Sequence In the Figuresmentioning

confidence: 99%

Dependence of the E. coli promoter strength and physical parameters upon the nucleotide sequence

Berezhnoy

Shckorbatov²

2005

J Zheijang Univ Sci B

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Abstract:The energy of interaction between complementary nucleotides in promoter sequences of E. coli was calculated and visualized. The graphic method for presentation of energy properties of promoter sequences was elaborated on. Data obtained indicated that energy distribution through the length of promoter sequence results in picture with minima at -35, -8 and +7 regions corresponding to areas with elevated AT (adenine-thymine) content. The most important difference from the random sequences area is related to -8. Four promoter groups and their energy properties were revealed. The promoters with minimal and maximal energy of interaction between complementary nucleotides have low strengths, the strongest promoters correspond to promoter clusters characterized by intermediate energy values.

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“…The consensus DNA sequence of the Ϫ10 region is 5Ј-TATAAT-3Ј in the nontemplate strand, with the first T being at the Ϫ12 position (1). To elucidate the molecular procedures of base unpairing during open complex formation, altered forms of DNA (depurinated, nicked, forked, or with single-stranded gap) in the Ϫ10 region were used as templates (10,(14)(15)(16)(17)(18)(19). These studies suggested that base unpairing involves an initial distortion or localized melting event around Ϫ11 (16,20).…”

Section: U Nder Ordinary Physiological Conditions Transcription Inmentioning

confidence: 99%

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

Lim

Lee

Roy

et al. 2001

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

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Isomerization of a closed to open complex of a promoter upon RNA polymerase binding involves base unpairing at the ؊10 region. After potassium permanganate sensitivity of unpaired thymine residues, we studied base unpairing at the ؊10 region during isomerization upon RNA polymerase binding at the P1 and P3 promoters of the gal operon. Substitution of adenine by 2-amino purine (2-AP) at the invariable A⅐T base pair at the ؊11 position of P1 and P3 prevented unpairing not only at that position but also at the other downstream positions, suggesting a ''master'' role of the adenine base at ؊11 of the template strand in overall base unpairing. 2-AP at ؊11 did not inhibit the formation of RNA polymerase⅐promoter complex and subsequent isomerization of the polymerase. Substitution of adenine by 2-AP at several other positions did not affect thymine unpairing. Changing the position of the amino group from C6 in adenine to C2 in 2-AP is mutational only at the master switch position, ؊11. U nder ordinary physiological conditions, transcription inEscherichia coli is executed by the RNA polymerase holoenzyme comprising ␣ 2 ␤␤Ј 70 subunits. Transcription initiation follows specific binding of RNA polymerase to the promotor. The initial binary complex (closed complex) undergoes structural changes (isomerization) in both proteins and DNA to make an open complex that is competent for subsequent templatedependent polymerization of ribonucleotides (1, 2). Whether isomerization of RNA polymerase and DNA occurs simultaneously or one after the other is unknown. The 70 subunit plays a key role in recognizing and making specific contacts with base pairs at the Ϫ35 and Ϫ10 regions of the promoter (3). In the open complex, DNA at the Ϫ10 region presumably becomes singlestranded for RNA polymerase to start reading the exposed template strand and polymerizing ribonucleotides (4-10). The bases in the nontemplate strand make specific interactions with amino acid residues of 70 (11). The consequence of these interactions is believed to stabilize the unstable nature of the unpaired DNA region (11-13). The consensus DNA sequence of the Ϫ10 region is 5Ј-TATAAT-3Ј in the nontemplate strand, with the first T being at the Ϫ12 position (1). To elucidate the molecular procedures of base unpairing during open complex formation, altered forms of DNA (depurinated, nicked, forked, or with single-stranded gap) in the Ϫ10 region were used as templates (10,(14)(15)(16)(17)(18)(19). These studies suggested that base unpairing involves an initial distortion or localized melting event around Ϫ11 (16,20). In this paper, we provide results by using intact duplex DNA to demonstrate that the adenine residue itself at the position Ϫ11 of the nontemplate strand plays a master role in the initiation of base unpairing. Materials and MethodsMaterials. E. coli RNA polymerase holoenzyme was purchased from Epicentre Technologies (Madison, WI). cAMP receptor protein (CRP), purified to 98% homogeneity by FPLC (Amersham Pharmacia) from an E. coli strain carrying the crp gen...

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In vitrotranscription close to the melting point of DNA: analysis ofThermotoga maritimaRNA polymerase—promoter complexes at 75°C using chemical probes

Cited by 24 publications

References 25 publications

Promoter opening via a DNA fork junction binding activity

Promoter opening via a DNA fork junction binding activity

Dependence of the E. coli promoter strength and physical parameters upon the nucleotide sequence

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

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