Conformational changes of <i>Escherichia coli</i> RNA polymerase on binding of templates

Stender, Walter

doi:10.1016/0014-5793(79)81249-1

Cited by 7 publications

(2 citation statements)

References 8 publications

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“…In the present study, monoclonal antibodies raised against the purified ß and ß' subunits of the E. coli RNA polymerase were used as site-specific reagents, Previous studies using antibody preparations raised against the individual subunits demonstrated that such preparations could inhibit in vitro transcription by RNA polymerase (Fukuda et al, 1977;Gragerov & Nikiforov, 1980;Slender, 1981). Anti-subunit polyclonal antibodies have been used by Slender (1979) to demonstrate conformational changes in RNA polymerase. Although subunit specific, polyclonal antibodies consist of a heterogeneous population of immunoglobulins able to crossroad with many of the antigenic determinants present on the subunit.…”

Section: Discussionmentioning

confidence: 99%

Characterization of effects of anti-.beta. and anti-.beta.' monoclonal antibodies on the activity of the RNA polymerase from Escherichia coli

Rockwell¹,

Beasley²,

Krakow³

1985

Biochemistry

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Monoclonal antibodies directed against antigenic determinants on the beta and beta' subunits of the Escherichia coli RNA polymerase were characterized by using d(A-T)n-directed transcription assays. Antibodies were prepared by using purified subunits as immunogens, and seven anti-beta and five anti-beta' monoclonal antibodies were generated. Inhibitory anti-beta monoclonal antibodies were found to affect RNA polymerase during synthesis of r(A-U)n, abortive initiation of pApU and UpApU, and elongation by preformed ternary complexes. A comparative enzyme study of r(A-U)n synthesis showed the core polymerase to be more sensitive to inhibition by the anti-beta monoclonal antibody than was the holoenzyme. In contrast, the inhibition effected by the anti-beta' monoclonal antibody was found to be 90% or greater for each of the d(A-T)n-directed assays used. The different inhibitory patterns exhibited by the anti-beta and anti-beta' monoclonal antibodies suggest that the beta and beta' subunits engage in different roles during transcription. Kinetic analysis of the abortive initiation reaction in the presence and absence of the inhibitory antibodies resulted in distinctive but complex modes of inhibition. Inhibition by the anti-beta monoclonal antibody 210E8 was noncompetitive with regard to UTP and competitive for UpA incorporation; at increased UpA concentration, the inhibition was completely reversed. Inhibition of the abortive synthesis of UpApU by the anti-beta' monoclonal antibody 311G2 was noncompetitive with regard to both UpA and UTP incorporation. When the preformed ternary elongation complex was used, inhibition by the anti-beta monoclonal antibody was mixed with regard to the ribonucleoside triphosphate substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 99%

Characterization of effects of anti-.beta. and anti-.beta.' monoclonal antibodies on the activity of the RNA polymerase from Escherichia coli

Rockwell¹,

Beasley²,

Krakow³

1985

Biochemistry

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“…Upon binding, the holoenzyme loses some secondary (Levine et al, 1980) and possibly higher order (Slender, 1979) structure and also monomerizes from the predominantly dimeric form in free solution (Krakow & von der Helm, 1970). It has been postulated that an RNAP molecule may possess more than one binding site for DNA (Reisbig et al, 1981;Park et al, 1980).…”

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confidence: 99%

Rotational diffusion of Escherichia coli RNA polymerase free and bound to deoxyribonucleic acid in nonspecific complexes

Austin¹,

Karohl²,

Jovin³

1983

Biochemistry

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We have studied the rotational diffusion of Escherichia coli RNA polymerase free in solution and bound nonspecifically to DNA fragments. The rotational motion was measured by the decay in anisotropy of the triplet-triplet absorption by using as probes either the liganded enzyme inhibitor Rose Bengal or eosin 5'-isothiocyanate conjugated to the protein. The time resolution extended from 10 ns to 1 ms. Free RNA polymerase (holoenzyme) at high salt concentration (1 M NaCl) is monomeric and diffuses at 5 degrees C with a rotational correlation time of 0.66 microseconds, corresponding to an equivalent hydrodynamic sphere with a radius of 7.4 nm. These values and the known molecular weight are most compatible with a nonspherical shape, e.g., an oblate ellipsoid with an axial ratio of about 3. In 0.1 M NaCl, the holoenzyme is dimeric and has a rotational correlation time of 2 microseconds. The decay of anisotropy is at least biexponential upon binding RNA polymerase to calf thymus DNA or to poly[d(A-T)]. The fast component with half of the amplitude has decay kinetics comparable to those seen with the free monomeric enzyme. The slow component has a rotational correlation time of about 14 microseconds and is independent of DNA chain length in the range greater than 180 base pairs. Both rotational correlation times decrease with temperature, and the relative amplitudes change such that the faster component dominates at higher temperature. The rotational relaxation of the enzyme-DNA complexes is discussed in terms of alternative models involving rigid rod-sphere diffusion, conformational changes in the enzyme and/or DNA, sliding motions of the protein along the DNA, and torsional-bending motions of DNA envisioned as a deformable rod.

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The structure and mechanism of action of bacterial DNA-dependent RNA polymerase

1981

Progress in Biophysics and Molecular Biology

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Conformational changes of Escherichia coli RNA polymerase on binding of templates

Cited by 7 publications

References 8 publications

Characterization of effects of anti-.beta. and anti-.beta.' monoclonal antibodies on the activity of the RNA polymerase from Escherichia coli

Characterization of effects of anti-.beta. and anti-.beta.' monoclonal antibodies on the activity of the RNA polymerase from Escherichia coli

Rotational diffusion of Escherichia coli RNA polymerase free and bound to deoxyribonucleic acid in nonspecific complexes

The structure and mechanism of action of bacterial DNA-dependent RNA polymerase

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