Studies of the binding of Escherichia coli RNA polymerase to DNA

Hinkle, David C.; Chamberlin, Michael J.

doi:10.1016/0022-2836(72)90531-1

Cited by 444 publications

(201 citation statements)

References 38 publications

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“…However, a role in termination has not previously been excluded. In particular sigma might be required to catalyse dissociation of core enzyme from the terminator DNA following RNA release, since Hinkle and Chamberlin have shown that addition of this subunit accelerates the dissociation of T7 DNA/core polymerase complexes in general (31,37).…”

Section: Concluding Discussionmentioning

confidence: 99%

Termination of transcription of the coliphage T7 “early” operon in vitro: slowness of enzyme release, and lack of any role for sigma

O’Hare

Hayward

1981

Nucl Acids Res

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The leftmost portion of the coliphage T7 genome is transcribed by the RNA polymerase Escherichia coli immediately after infection.This "early" operon is delineated by three promoters on the left, and a transcriptional terminator on the right.The terminator is efficient both in vivo, and with highly purified RNA polymerase in vitro.We have studied termination in vitro, using synchronously initiated transcription reactions with low enzyme:DNA ratios.We show that recognition of the stop signal and release of RNA product are relatively rapid.Dissociation of the enzyme from the DNA is quite slow, and probably rate-limiting for re-cycling of the polymeras.It is well established that the sigma subunit of RNA polymerase is required for specific initiation, but redundant during RNA elongation. By exploiting antisigma antiserum we have abtained evidence that sigma is also redundant during all steps of termination in vitro.

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Section: Concluding Discussionmentioning

confidence: 99%

Termination of transcription of the coliphage T7 “early” operon in vitro: slowness of enzyme release, and lack of any role for sigma

O’Hare

Hayward

1981

Nucl Acids Res

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“…DNA-binding assays were performed as described by Hinkle and Chamberlin [17] using calf thymus or T7…”

Section: Dna-binding Assaysmentioning

confidence: 99%

“…We investigated the effect of the antibody on enzyme-DNA complex formation using the property of the complex to be retained on nitrocellulose filters [17]. Using this kind of experiment, Carrol and Stollar [9] have shown that inhibition of calf thymus RNA polymerase B by a specific monoclonal antibody is due to the fact that the antibody, when fixed onto the enzyme, prevents the enzyme-DNA complex formation.…”

Section: Reactivity Of A14 and A17 Monoclonal Antibodies Towards Eukamentioning

confidence: 99%

A monoclonal antibody directed against a small subunit of RNA polymerase B blocks the initiation step

Vilamitjana¹,

Barreau²

1987

Eur J Biochem

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Monoclonal antibodies directed against RNA polymerase B of the fungus Podospora were probed for their inhibitory effect on enzyme activity in vitro. From nine antibodies directed against various subunits of the enzyme only two of them totally inhibit RNA polymerase B activity in a non-selective transcription system. Enzymes A and C are not inhibited at all. The two antibodies recognize the same small subunit (B11) of Podosporu enzyme B. One of these antibodies has been used as a specific inhibitor to study the function of the B11 subunit. Enzyme activity is partially protected from the inhibition when the enzyme is previously incubated with the template. The antibody does not interfere with enzyme-DNA complex formation but blocks the initiation process in a transcription system using a dinucleotide as initiator. Elongation of RNA chains does not appear to be affected. Also the antibody cross-reacts with yeast and calf thymus RNA polymerases B and has some, but less, effect on initiation by these enzymes. It is suggested the antigenic site corresponding to this antibody may overlap the catalytic initiation site on the native form of RNA polymerase B.In eukaryotes the three classes of genes encoding rRNA, heterogenous nuclear RNA, and 5s RNA and tRNA are transcribed by three distinct RNA polymerases termed A (or I), B (or 11) and C (or 111) [l, 21. They have so complex a subunit structure that very little is known about the part played by the individual subunits in enzyme function.Few specific RNA polymerase inhibitors are available and genetic studies failed to give any information. Some of the subunits (possibly the common subunits of the three enzymes) may be responsible for the basic steps of the transcription reaction while the others may be implicated in the specificity of the transcription or in the regulation of transcriptional activity during differentiation processes. Specific probes for each subunit will greatly help in investigating these complex enzymes.Antibodies directed against the different subunits of yeast RNA polymerases [3] have allowed the precise structural characterization of yeast transcriptional apparatus. Functional studies have shown that most of the antibodies have an inhibitory effect in a non-specific transcription system, arguing in favour of the idea that all the subunits take part in the work of the enzyme. However, these studies have been carried out with polyspecific antibodies. For such functional studies more specific probes may be obtained with the use of monoclonal antibodies. calf thymus [8, 91. Few of these antibodies exhibit inhibitory effects in a non-specifi; transcription system. This does not exclude the possibility that they may inhibit specific transcription in appropriate systems as shown for a monoclonal antibody directed against RNA polymerase B from calf thymus [lo].We previously reported the characterization of nine monoclonal antibodies directed against RNA polymerase B from the fungus Podospora [ll]. Here we report the use of one of these antibodies as a spec...

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“…The first step in initiation is promoter recognition and binding by RNA polymerase. Nitrocellulose filter retention has been used to evaluate DNA binding at p R , and the complexes retained are open complexes (6,7,16,19,20). Holoenzyme (1 nM) was incubated with a 32 P-5Ј-end-labeled DNA fragment containing p R (0.1 nM).…”

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confidence: 99%

Effects of Amino Acid Substitutions at Conserved and Acidic Residues within Region 1.1 of Escherichia coli ς ⁷⁰

2000

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Amino acid substitutions in Escherichia coli 70 were generated and characterized in an analysis of the role of region 1.1 in transcription initiation. Several acidic and conserved residues are tolerant of substitution. However, replacement of aspartic acid 61 with alanine results in inactivity caused by structural and functional thermolability.Core RNA polymerase (␣ 2 ␤␤Ј) requires the variable specificity subunit, sigma ( ), to direct promoter-dependent transcription (1,3,4,12,18,22,23,26). Following promoter binding, holoenzyme (␣ 2 ␤␤Ј ) progresses through several intermediate complexes, en route to a stable initiated open complex (2,14).factor has been implicated in stages of initiation beyond promoter recognition (8,9,13,15,17). Recently, we showed that the conserved amino terminal domain (region 1) of Escherichia coli 70 is important for the process of strand melting and initiated complex formation at the p R promoter (24).Region 1 is unique to the primary factors, yet little is known of its function. Deletion of region 1.1 (amino acids 1 to 100) from 70 has two major consequences for holoenzyme. The first is inefficient progression from the closed to the strand-separated open complex. This can be overcome by increasing the time allowed for formation of holoenzyme-promoter complexes and is lessened by addition of region 1.1 in trans. The second and more deleterious effect is impaired transition from the strand-separated open complex to a stable initiated complex (RP init D]), as well as a high degree of acidity (40%) within the segment from amino acids 50 to 75 (24). Here, we test whether alterations at these conserved positions or in the overall acidity of the region influence initiation by holoenzyme (E ).Site-directed mutagenesis (10) and the Expand high fidelity PCR system (Boehringer Mannheim) were used to create substitutions at positions 52, 53, 55, 61, 57, 58, 63, 64, and 69 (Table 1). rpoD was mutagenized in M13 phage (10) and amplified with oligonucleotides that incorporated restriction sites at the 5Ј and 3Ј ends of the fragment. The restricted fragments were ligated into pQE30-T (24). PCR mutagenesis was used to amplify a fragment corresponding to the 3Ј end of the rpoD gene with a 5Ј mutagenic oligonucleotide and a 3Ј oligonucleotide that incorporated a restriction site. A concurrent round of amplification included a 5Ј oligonucleotide complementary to the 5Ј end of rpoD and a 3Ј oligonucleotide with complementarity to an internal segment of rpoD, downstream from the genetic alteration(s). The 5Ј and 3Ј PCR fragments were mixed, and the full-length mutagenized rpoD gene was amplified, gel isolated, digested, ligated into pQE30-T, transformed into E. coli XL1 Blue (Stratagene), and sequenced to confirm the changes. The plasmids were transformed into E. coli 19284 (rpoD800, W3110 srl::Tn10 recA lacI q ) to test for function in vivo (24). Transformation mixtures were split and spread onto Luria-Bertani plates containing ampicillin (100 mg/ml), kanamycin (30 mg/ml), and 2% glucose and then inc...

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Studies of the binding of Escherichia coli RNA polymerase to DNA

Cited by 444 publications

References 38 publications

Termination of transcription of the coliphage T7 “early” operon in vitro: slowness of enzyme release, and lack of any role for sigma

Termination of transcription of the coliphage T7 “early” operon in vitro: slowness of enzyme release, and lack of any role for sigma

A monoclonal antibody directed against a small subunit of RNA polymerase B blocks the initiation step

Effects of Amino Acid Substitutions at Conserved and Acidic Residues within Region 1.1 of Escherichia coli ς ⁷⁰

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Studies of the binding of Escherichia coli RNA polymerase to DNA

Cited by 444 publications

References 38 publications

Termination of transcription of the coliphage T7 “early” operon in vitro: slowness of enzyme release, and lack of any role for sigma

Termination of transcription of the coliphage T7 “early” operon in vitro: slowness of enzyme release, and lack of any role for sigma

A monoclonal antibody directed against a small subunit of RNA polymerase B blocks the initiation step

Effects of Amino Acid Substitutions at Conserved and Acidic Residues within Region 1.1 of Escherichia coli ς 70

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Effects of Amino Acid Substitutions at Conserved and Acidic Residues within Region 1.1 of Escherichia coli ς ⁷⁰