Small‐angle x‐ray study of DNA‐dependent RNA polymerase subunit α<sub>2</sub> from <i>Escherichia coli</i>

Meisenberger, Otto; Pilz, Ingrid; Heumann, Hermann

doi:10.1016/0014-5793(80)81045-3

Cited by 15 publications

(10 citation statements)

References 9 publications

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“…Core enzyme and holoenzyme were purified as in [3,4]. The homodispersity, purity and activity of hdoenzyme were checked as in [3].…”

Section: Preparation Of Rna Polymerasementioning

confidence: 99%

“…Models of aggregated RNA polymerase were built by different arrangements of models of monomeric RNA poly- merase holoenzyme and core enzyme. These models were proposed in [4,7]. This method does not allow, a direct determination of the shape of the aggregates, but models which fit the experimental curves within the experimental error can be considered to have a high degree of reliability.…”

Section: Shapementioning

confidence: 99%

“…Core enzyme and holoenzyme were purified as in [3,4]. The homodispersity, purity and activity of hdoenzyme were checked as in [3]. For the measuremerits the proteins were dialySed overnight against a buffer containing 0.05 M Tris-HCl (pH 7.5) and 10-3 M mercaptoethanol.…”

Section: Preparation Of Rna Polymerasementioning

confidence: 99%

“…A model of DNA -dependent RNA polymerase from E. coli has been developed on the basis of neutron and X-ray small angle scattering measurements [1][2][3][4][5][6][7]. These studies were performed in buffers of high ionic strength, in which the enzyme is monomerle.…”

Section: Introductionmentioning

confidence: 99%

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Small‐angle X‐ray study of aggregates of DNA‐dependent RNA polymerase from Escherichia coli

Heumann¹,

Meisenberger²,

Pilz³

1982

FEBS Letters

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“…Core enzyme and holoenzyme were purified as in [3,4]. The homodispersity, purity and activity of hdoenzyme were checked as in [3].…”

Section: Preparation Of Rna Polymerasementioning

confidence: 99%

Section: Shapementioning

confidence: 99%

Section: Preparation Of Rna Polymerasementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Small‐angle X‐ray study of aggregates of DNA‐dependent RNA polymerase from Escherichia coli

Heumann¹,

Meisenberger²,

Pilz³

1982

FEBS Letters

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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.…”

mentioning

confidence: 99%

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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Small-angle X-ray study of DNA-dependent RNA polymerase fromEscherichia Coli

Meisenberger

Pilz

Heumann

1981

Colloid Polymer Sci

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Small‐angle x‐ray study of DNA‐dependent RNA polymerase subunit α₂ from Escherichia coli

Cited by 15 publications

References 9 publications

Small‐angle X‐ray study of aggregates of DNA‐dependent RNA polymerase from Escherichia coli

Small‐angle X‐ray study of aggregates of DNA‐dependent RNA polymerase from Escherichia coli

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

Small-angle X-ray study of DNA-dependent RNA polymerase fromEscherichia Coli

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Small‐angle x‐ray study of DNA‐dependent RNA polymerase subunit α2 from Escherichia coli

Cited by 15 publications

References 9 publications

Small‐angle X‐ray study of aggregates of DNA‐dependent RNA polymerase from Escherichia coli

Small‐angle X‐ray study of aggregates of DNA‐dependent RNA polymerase from Escherichia coli

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

Small-angle X-ray study of DNA-dependent RNA polymerase fromEscherichia Coli

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Small‐angle x‐ray study of DNA‐dependent RNA polymerase subunit α₂ from Escherichia coli