Small‐angle X‐ray study of aggregates of DNA‐dependent RNA polymerase from <i>Escherichia coli</i>

Heumann, Hermann; Meisenberger, Otto; Pilz, Ingrid

doi:10.1016/0014-5793(82)80459-6

Cited by 9 publications

(10 citation statements)

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“…It has been reasonably well documented that both holo and core RNA polymerase exist in different states of aggregation as a function of salt concentration (Richardson, 1966;Stevens et al, 1966;Burgess, 1969a;Berg & Chamberlin, 1970;Shaner et al, 1982;Heumann et al, 1982).…”

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confidence: 98%

Self-association of Escherichia coli DNA-dependent RNA polymerase core enzyme

Harris¹,

Williams²,

Lee³

1995

Biochemistry

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The extent of self-association of Escherichia coli DNA-dependent RNA polymerase core enzyme has been investigated by velocity sedimentation as a function of both NaCl and protein concentrations. The core enzyme, existing as essentially monomeric species having a sedimentation coefficient of 13.1 S at NaCl concentrations greater than 0.2 M, undergoes reversible self-association at lower salt concentrations. Estimates for the stoichiometry of association and equilibrium constants of reaction were determined from the effect of protein concentration on the weight-average sedimentation coefficient measured at different NaCl concentrations. Data analysis by a nonlinear curve-fitting procedure indicated that protein self-association is best described by a sequential model characterized by weaker association constants for each additional step of oligomerization, and any model that involves cooperative formation of oligomeric species can be excluded. These findings are at variance with the conclusion of a previous study [Shaner, S. L., Platt, D. M., Wensley, C. G., Yu, H., Burgess, R. R., & Record, M. T. (1982) Biochemistry 26, 5539-5551] which suggested that core RNA polymerase exists in equilibrium between monomeric and tetrameric forms of the enzyme and excluded the existence of intermediate species. Simulation of sedimentation velocity boundary and gradient profiles are used to assess the validity of both models of association of core protein. It was clear that had the core enzyme undergone a cooperative monomer<-->tetramer mode of association, then bimodality would have been observed in the derivative tracings of the sedimentation boundary under these experimental conditions. Nevertheless, no such observation was reported by Shaner et al. and this study. The sequential model favored by the results of this study is consistent with the proposed model resulted from a small-angle X-ray study [Heumann, H, Meisenberger O., & Pilz, I. (1982) FEBS Lett. 138, 273-276]. Further analysis of the data by the Wyman linked-function relationship [Wyman, J. (1964) Adv. Protein Chem. 19, 223-286] implies that core enzyme monomer loses approximately three counterions per contact upon association to higher oligomeric species.

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confidence: 98%

Self-association of Escherichia coli DNA-dependent RNA polymerase core enzyme

Harris¹,

Williams²,

Lee³

1995

Biochemistry

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“…The susceptibility of the polymerase subunits to attack by proteases (17)(18)(19) has been investigated in both the isolated enzyme and in binary complexes with DNA. In addition to these chemical studies, physical methods such as smallangle x-ray scattering (20)(21)(22)(23) and neutron scattering (24, 25) have provided information on the size and shape of the enzyme.…”

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confidence: 99%

“…Samples were withdrawn from each reaction mixture prior to photolysis for analysis on polyacrylamide gels. Aliquots (10 t'l) were added to 20 Ml of saturated urea and 20 Ml of saturated phenylmercuric acetate in 0.1% NaDodSO4. These were allowed to hydrolyze to oligonucleotide 5'-monophosphates overnight in the dark at room temperature.…”

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confidence: 99%

“…All transcription reactions were carried out in dim light. Transcription complexes containing radiolabeled RNA chains of [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] 3'-O-methyl-CTP at 0.5 mM, or 3'-O-methyl-GTP at 0.25 mM). The buffer, DNA, RNA polymerase, and N3RSpApU were preincubated at 370C for 5 min before addition of nucleotides.…”

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“…Transcription mixtures were transferred to 6 x 20 mm borosilicate glass tubes and irradiated for 2 min in a Rayonet type RS photochemical reactor (A > 300 nm). The reaction mixtures containing RNAs <20 bases long were treated with 20 Ml of buffer B and 10 Ml of 1 M dithiothreitol, and allowed to sit for 1 hr at room temperature in the dark to reduce any remaining azide. The samples then were loaded onto 40 cm X 0.75 mm NaDodSO4/urea (31) step gradient gels in buffer C (32 cm of 3.5% acrylamide as upper layer; 6 cm of 7% acrylamide as lower layer).…”

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Topography of transcription: path of the leading end of nascent RNA through the Escherichia coli transcription complex.

Hanna¹,

Meares²

1983

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

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A cleavable dinucleotide photoaffinity reagent was prepared and used to map the path of the leading end of the RNA transcript across the surface of Escherichia coli RNA polymerase/ T7 DNA transcription complexes. By using 5'-(4-azidophenacylthio)phosphoryladenylyl(3'-5')uridine, transcription was specifically initiated at the Al promoter of bacteriophage T7 DIll or D123 DNA. Transcription complexes containing radiolabeled RNA chains of various lengths (4-116 nucleotides) were prepared, and the 5' end of the RNA transcript was then covalently attached to the nearby polymerase subunits or DNA by irradiation with UV light. The photoaffinity-labeled enzyme subunits and DNA were separated, the radiolabeled RNAs were cleaved from each, and the lengths and sequences of RNA attached to each component were determined. The leading end of RNA chains up to 12 bases long was found to label the DNA and the ,3 and /3' subunits of RNA polymerase, with more than 90% of the label going to the DNA. When the RNA transcript reached 12 bases in length, the 5' end diverged from the DNA and only the /8 and /3' enzyme subunits were labeled thereafter. These two subunits were heavily labeled by RNA chains 12 to as many as 94 bases long. No signifitant labeling of the a subunit occurred. The a subunit was not labeled by RNAs longer than the trinucleotide.DNA-dependent RNA polymerases play a key role in gene expression. Escherichia coli RNA polymerase, the most extensively studied, contains five major subunits and has a Mr of approximately 454,000 (1). The "core" enzyme consists of subunits A3' (Mr, 160,000), /3 (Mr, 151,000), and two a subunits (Mr,36,000) and is capable of RNA elongation after transcription has been initiated. The holoenzyme contains the core plus the dissociable subunit or (Mr, 70,000) have been determined. Our method involves the use of a cleavable photoaffinity probe, containing both a photosensitive azide moiety for covalent attachment and a selectively cleavable S-P bond for subsequent removal of the attached oligonucleotide. We chose to prepare the dinucleotide photoaffinity reagent we used (Fig. 1) for three reasons: dinucleotides initiate transcription much more efficiently than mononucleotides (26); the aromatic azide moiety produces a chemically reactive, relatively nonselective nitrene (27) upon illumination with wavelengths greater than 300 nm; and the S-P bond was found to be stable under various conditions, although it could be cleanly hydrolyzed in the presence of organomercurials.At the initiation of transcription, the photoaffinity probe is incorporated into the leading (5') end of a nascent RNA chain. Because addition of nucleoside triphosphates occurs at the 3' end of the growing RNA chain in the elongation site of the enzyme, the 5' end must move out over the surface of the enzyme-DNA complex as transcription proceeds. Transcription complexes containing RNA chains of almost every length from 4 to 116 nucleotides have been prepared by using RNA chain terminators that substitute for ATP, CTP...

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