Template-Induced Dissociation of Ribonucleic Acid Polymerase<sup>*</sup>

Da, Smith; Am, Martinez; Rl, Ratliff; Williams, Dennis; Fn, Hayes

doi:10.1021/bi00862a012

Cited by 37 publications

(10 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, the failure of three nucleoside triphosphates to cause significant amount of af release suggests the a does not release during the initiation process itself, but after the RNA chains have reached a certain length. In accord with this observation is the finding by Krakow and Fronk (26) (2), the decrease in 0 might reflect the template-induced dissociation of RNA polymerase dimer into monomers (27). If the latter is the case, the observed ) value of 508 nsec suggests that the dissociated monomeric enzyme is still bound to the DNA template because the value of 4 for a free monomeric DNS-a-core enzyme complex would be about 300 nsec.…”

Section: Resultssupporting

confidence: 76%

Sigma cycle during in vitro transcription: demonstration by nanosecond fluorescence depolarization spectroscopy.

Wu¹,

Yarbrough²,

Hillel³

et al. 1975

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

View full text Add to dashboard Cite

present. After a was released, addition of core polymerase with rifampicin reduced the free a to less than 15%, indicating that the released a was accessible to the added core enzyme. Thus these studies have provided physical evidence for the a cycle during in vitro transcription. The transcription of a bacterial genome is primarily mediated by a single RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6). The purified Escherichia coli RNA polymerase holoenzyme has a subunit structure of a2If'a, with molecular weights for the subunits of 40,000, 155,000, 165,000, and 90,000, respectively (1, 2). The holoenzyme can be reversibly dissociated to yield the core enzyme (a2o') and the a subunit. Like the holoenzyme, core polymerase is catalytically active, but differs in transcribing native double-stranded DNA nonspecifically and inefficiently (3, 4).The a subunit by itself has no catalytic function. Its role in gene transcription is 2-fold. (a) The a specificity: a promotes specific initiation of RNA chains that yields asymmetric transcription resembling in vvo RNA products (1). (b) The a cycle: a stimulates RNA synthesis by increasing the rate of initiation through its catalytic reuse by core polymerase (5).Based on their in tdtro transcriptional studies, Travers and Burgess (5) first proposed the following a cycle: a initially forms a complex with core polymerase, which is able to bind to promotor sites on DNA and to initiate specific RNA synthesis. During or after initiation, a is released from the enzyme-DNA complex and may then be reused by another core polymerase molecule to initiate a new RNA chain.Evidence for the physical separation of a from the enzyme-DNA complex after RNA chain initiation has been provided by use of polyacrylamide gel electrophoresis for Azotobacter and E. coli RNA polymerases (6, 7), or by use of sucrose gradient centrifugation for Pseudomonas RNA polymerase (8). However, the evidence is not conclusive because electrical or centrifugal force used could influence the interaction between a and core polymerase. Even if the effect of the external forces is negligible, these studies only indicate a weakening of the a-core polymerase interaction during transcription, resulting in the subsequent separation of a from core polymerase by the electric field or centrifugal force, and do not prove the physical release of a due to RNA synthesis.This communication presents a direct demonstration by nanosecond fluorescence depolarization spectroscopy of the physical release of a during transcription. This technique measures the rate of molecular rotation (Brownian rotational diffusion) and thereby provides information concerning the sizes, shapes, and aggregation states of macromolecules (9, 10) MATERIALS AND METHODS RNA polymerase holoenzyme was purified from cells of E. coli B as described previously (11). The purity of the enzyme was >95% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The a subunit and core polymerase were...

show abstract

Section: Resultssupporting

confidence: 76%

Sigma cycle during in vitro transcription: demonstration by nanosecond fluorescence depolarization spectroscopy.

Wu¹,

Yarbrough²,

Hillel³

et al. 1975

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

View full text Add to dashboard Cite

show abstract

“…RNA polymerase holoenzyme exists in dimeric or monomeric form depending upon ionic strength [23,24]. To probe the effect of these structural differences on the reaction with fluorescamine 1 M NaCl was added to the Hepes buffer (designated high ionic strength).…”

Section: High and Low Salt Conditionsmentioning

confidence: 99%

“…1, addition of monomeric subtrate diminished the effect. In the presence of 1.3 M NaCl, under which condition RNA polymerase does not bind to DNA [24], poly[d(A-T)] no longer influenced the reaction.…”

Section: Inactivation Of Rna Polymerase By Fluorescarninementioning

confidence: 99%

See 1 more Smart Citation

The Functional and Fluorescence Properties of Escherichia coli RNA Polymerase Reacted with Fluorescamine

Damjanovich¹,

Bähr

Jovin

1977

European Journal of Biochemistry

View full text Add to dashboard Cite

1. Fluorescamine (4-phenylspiro[furan-2,(3) 1 '-phthalan]-3,3'-dione) reacts rapidly with Escherichia coli RNA polymerase and produces a fluorescent derivative which is inactivated to an extent dependent upon reagent concentration. Excess fluorescamine is rapidly hydrolysed. Reaction is with &-amino groups of lysine residues in all subunits as revealed by gel electrophoresis and fluorescence scanning.2. The extent of inactivation and fluorescence yield are diminished in the presence of added template, a finding which provides evidence for the existence of reactive and essential amino groups which can be at least partially shielded by DNA in the binary complexes. The relative decrease of fluorescence is greatest in the PP' subunits. Holoenzyme and core enzyme show essentially the same behavior.3. The inactivation of activity by fluorescamine is primarily at the level of initiation. Template binding and chain propagation are less affected.4. The enzyme derivatized by fluorescamine shows an intense fluorescence with a peak at 490 nm and an excitation maximum at 390 nm. The fluorescence lifetime is in the range of 3-8 ns and the emission is highly polarized. In reactions carried out at high ionic strength the fluorescence yield is approximately double that at low ionic strength and insensitive to the presence of template.5. Energy transfer is observed between the derivatized enzyme as donor and ethidium bromide as acceptor in the presence of template to which both the enzyme and intercalating dye are bound. The transfer efficiency is a function of the relative concentrations and of the conditions of reaction with fluorescamine. An average transfer distance of approx. 4-5 nm has been calculated suggesting a close proximity between bound polymerase and helical regions of the template.The initial event in the transcription of genetic information is the specific interaction between RNA polymerase and template DNA [l -51. Although the process of specific site selection has been studied extensively (for reviews see [3][4][5]), relatively little information exists regarding the molecular interrelationships within the binary complexes.The structural and functional properties of the procaryotic RNA polymerases have been rather well elucidated [5]. Two of the three subunits constituting the core enzyme (a$P'), p and p', have been identified as the primary loci for template binding and initiation respectively, and the additional subunit c, which forms part of the holoenzyme, is responsible for the specificity of promoter recognition. A tentative model Enzyme. DNA-dependent RNA polymerase (EC 2.7.7.6).has been presented for the topographical interrelationships between the various subunits [6]. Numerous studies using chemical modification and affinity labelling (mainly of the -SH and -NH2 groups) have sought to identify the important amino acid side-groups responsible for substrate and template binding and for catalysis. In particular, it has been possible to label the P subunit covalently (and inhibit the enzyme) with the pseud...

show abstract

“…One explanation might be that the binding of DNA to one of the two binding sites of the dimer reduces the affinity of the site on the other monomer for DNA. I n this respect it would be of interest to determine whether a high DNA to enzyme ratio induces dissociation of the dimer as happens with the dimer of Esch,erichia coli RNA polymerase [29].…”

Section: Template Requirementsmentioning

confidence: 99%

Purification and Partial Characterisation of Rat‐Liver Nuclear DNA Polymerase

Haines¹,

Wickremasinghe

Johnston

1972

European Journal of Biochemistry

View full text Add to dashboard Cite

A method is described for the preparation of DNA polymerase purified about 800‐fold from rat liver nuclei. The yield of enzyme is about 140–200 μg from 200 g liver. Sodium dodecylsulphate‐polyacrylamide gel electrophoresis of the enzyme in the final step, shows a main band corresponding to a polypeptide of molecular weight of 29000 ± 3%. Sephadex G‐100 column chromatography indicates the enzyme to have an apparent molecular weight of approximately 60000 ± 2% at an ionic strength of 0.15, suggesting that the enzyme is a dimer as isolated. In 2 M NaCl, the apparent molecular weight is 42000. The enzyme prefers double‐stranded DNA templates but utilises most efficiently those activated by deoxyribonuclease I. It has the ability to carry out limited synthesis using only one deoxynucleoside‐5′‐triphosphate in the assay. The final preparation of DNA polymerase has nucleoside diphosphate kinase associated with it.

show abstract

Template-Induced Dissociation of Ribonucleic Acid Polymerase^*

Cited by 37 publications

References 14 publications

Sigma cycle during in vitro transcription: demonstration by nanosecond fluorescence depolarization spectroscopy.

Sigma cycle during in vitro transcription: demonstration by nanosecond fluorescence depolarization spectroscopy.

The Functional and Fluorescence Properties of Escherichia coli RNA Polymerase Reacted with Fluorescamine

Purification and Partial Characterisation of Rat‐Liver Nuclear DNA Polymerase

Contact Info

Product

Resources

About

Template-Induced Dissociation of Ribonucleic Acid Polymerase*

Cited by 37 publications

References 14 publications

Sigma cycle during in vitro transcription: demonstration by nanosecond fluorescence depolarization spectroscopy.

Sigma cycle during in vitro transcription: demonstration by nanosecond fluorescence depolarization spectroscopy.

The Functional and Fluorescence Properties of Escherichia coli RNA Polymerase Reacted with Fluorescamine

Purification and Partial Characterisation of Rat‐Liver Nuclear DNA Polymerase

Contact Info

Product

Resources

About

Template-Induced Dissociation of Ribonucleic Acid Polymerase^*