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Volumn 276, Issue 5316, 1997, Pages 1258-1260

Promoter recognition as measured by binding of polymerase to nontemplate strand oligonucleotide

Author keywords

[No Author keywords available]

Indexed keywords

DNA DIRECTED DNA POLYMERASE ALPHA;

EID: 0030915128     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5316.1258     Document Type: Article
Times cited : (151)

References (31)
  • 5
    • 1842385648 scopus 로고    scopus 로고
    • note
    • 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.
  • 6
    • 1842361629 scopus 로고    scopus 로고
    • note
    • 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.
  • 9
    • 1842379848 scopus 로고    scopus 로고
    • Qiagen, Chatsworth, CA
    • Qiagen, Chatsworth, CA.
  • 10
    • 1842280851 scopus 로고    scopus 로고
    • note
    • 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).
  • 16
    • 0027761839 scopus 로고
    • W. Ross et al., ibid. 262, 1407 (1993).
    • (1993) Science , vol.262 , pp. 1407
    • Ross, W.1
  • 17
    • 1842341192 scopus 로고    scopus 로고
    • 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
    • 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.
  • 20
    • 1842345982 scopus 로고    scopus 로고
    • note
    • 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.
  • 31
    • 1842313397 scopus 로고    scopus 로고
    • note
    • 70. Supported by a grant from NIH.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.