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1
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0002424825
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W. Reznikoff, Ed. Elsevier, New York
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R. Burgess et al., in RNA Polymerase and the Regulation of Transcription, W. Reznikoff, Ed. (Elsevier, New York, 1987), p. 3.
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(1987)
RNA Polymerase and the Regulation of Transcription
, pp. 3
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Burgess, R.1
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2
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0026755533
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A. J. Dombroski, W. A. Walter, M. T. J. Record, D. A. Siegele, C. A. Gross, Cell 70, 501 (1992).
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(1992)
Cell
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, pp. 501
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Dombroski, A.J.1
Walter, W.A.2
Record, M.T.J.3
Siegele, D.A.4
Gross, C.A.5
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5
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1842385648
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note
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2, 0.1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, and bovine serum albumin (100 μg/ml). The complexes were run on a 5% 0.5 × tris-borate EDTA native polyacrylamide gel at 4°C. The gel was imaged and radioactivity was measured on a Fuji BAS2000 or a Phosphorlmager (Molecular Dynamics). Bound oligo was defined as any radioactivity of less mobility than the free DNA, including the defined band at the protein position and radioactivity released during electrophoresis. The positions of core and holoenzyme were determined by silver staining of standards.
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6
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1842361629
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note
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2+-nitrilotriacetic acid agarose beads from Qiagen (9). The Q437H mutation was created by polymerase chain reaction (PCR) with a primer containing the mutation. The PCR product was then subcloned into the pET-28 vector containing wild-type sigma polypeptide. Clones containing the mutation were verified by sequencing. The sigma polypeptide was then purified exactly as the wild-type His-tagged sigma polypeptide. Holoenzyme was reconstituted by incubation of core enzyme for 30 min on ice with excess (five times greater) of the desired sigma factor.
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9
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1842379848
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Qiagen, Chatsworth, CA
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Qiagen, Chatsworth, CA.
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10
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1842280851
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note
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Oligos were synthesized at the Cornell DNA synthesis facility, radiolabeled as described (11) except that the reaction time was extended to 12 hours, and purified on an 8% polyacrylamide gel by a modification of the crush-and-soak method (11).
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11
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0004136246
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Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, ed. 2
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J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, ed. 2, 1989).
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(1989)
Molecular Cloning: A Laboratory Manual
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Sambrook, J.1
Fritsch, E.F.2
Maniatis, T.3
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13
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0025066083
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C. Waldburger, T. Gardella, R. Wong, M. M. Susskind, J. Mol. Biol. 215, 267 (1990).
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(1990)
J. Mol. Biol.
, vol.215
, pp. 267
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Waldburger, C.1
Gardella, T.2
Wong, R.3
Susskind, M.M.4
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15
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0029818631
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E. Nudler, E. Avetissova, V. Markovtsov, A. Goldfarb, Science 273, 211 (1996).
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(1996)
Science
, vol.273
, pp. 211
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Nudler, E.1
Avetissova, E.2
Markovtsov, V.3
Goldfarb, A.4
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16
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0027761839
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W. Ross et al., ibid. 262, 1407 (1993).
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(1993)
Science
, vol.262
, pp. 1407
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Ross, W.1
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17
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1842341192
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EMSA was performed as described above, except that the oligo concentration was 0.05 nM, and the polymerase concentration varied from 0 to 60 nM
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EMSA was performed as described above, except that the oligo concentration was 0.05 nM, and the polymerase concentration varied from 0 to 60 nM.
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18
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0024371540
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D. A. Siegele, J. C. Hu, W. A. Walter, C. A. Gross, J. Mol. Biol. 206, 591 (1989).
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(1989)
J. Mol. Biol.
, vol.206
, pp. 591
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Siegele, D.A.1
Hu, J.C.2
Walter, W.A.3
Gross, C.A.4
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20
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1842345982
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note
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For cross-linking, 20-μl binding reaction mixtures containing 6 nM oligo C and 60 nM RNA polymerase were exposed on ice for 10 min to 256-nm light from an ultraviolet transilluminator with the filter removed and analyzed on an SDS-8% polyacrylamide gel. The cross-linked species were visualized by autoradiography, and the proteins were localized by silver staining.
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22
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0025369026
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P. Schickor, W. Metzger, W. Werel, H. Lederer, H. Heumann, EMBO J. 9, 2215 (1990).
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(1990)
EMBO J.
, vol.9
, pp. 2215
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Schickor, P.1
Metzger, W.2
Werel, W.3
Lederer, H.4
Heumann, H.5
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24
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0025974528
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M. Buckle, J. Geiselmann, A. Kolb, H. Buc, Nucleic Acids Res. 19, 833 (1991).
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(1991)
Nucleic Acids Res.
, vol.19
, pp. 833
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Buckle, M.1
Geiselmann, J.2
Kolb, A.3
Buc, H.4
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31
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1842313397
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note
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70. Supported by a grant from NIH.
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