Elongin (SIII): A multisubunit regulator of elongation by RNA polymerase II

Science

Volumn 269, Issue 5229, 1995, Pages 1439-1443

  Aso, Teijiro ^a   Lane, William S ^b   Conaway, Joan Weliky ^a   Conaway, Ronald C ^a  

^a OKLAHOMA MEDICAL RESEARCH FOUNDATION   (United States)

^b HARVARD UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ELONGATION FACTOR; PROTEIN SUBUNIT; RNA POLYMERASE II;

AMINO ACID SEQUENCE; ARTICLE; GENE EXPRESSION; GENE ISOLATION; MULTIGENE FAMILY; PRIORITY JOURNAL; PROTEIN STRUCTURE; RNA TRANSLATION;

MAMMALIA;

EID: 0029084914     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.7660129     Document Type: Article

References (37)

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18. Approximately 300 pmol of Elongin A was isolated by reversed-phase HPLC (12). After reduction, S-carboxyamidomethylation, and digestion with trypsin, the resultant mixture was further fractionated by microbore HPLC. Optimal peptides were determined by differential absorbance of ultraviolet light and matrix-assisted laser desorption mass spectrometry (Lasermat; Finnigan-MAT, San Jose, CA). The peptides were then sequenced by automated Edman degradation (32).
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21. 4, 0.1 mM EDTA, and 10% (v/v) glycerol. The column was eluted at 1 ml/min with a 50-ml linear gradient from 0.1 to 0.8 M KCI in the same buffer. Fractions (1 ml) were collected. Elongin A eluted between 0.35 and 0.40 M KCI.
22. 2-terminal CGG) was coupled to maleimide-activated ovalbumin with use of an immunoject activated immunogen conjugation kit (Pierce) according to the manufacturer's instructions. The resultant conjugate (0.2 mg) was injected in complete Freund's adjuvant into a rabbit. The rabbit was boosted at 4-week intervals with 0.2 mg of conjugated peptide in complete Freund's adjuvant.
23. T. Aso, J. W. Conaway, R. C. Conaway, unpublished results.
24. Renaturation of the recombinant Elongin subunits was carried out essentially as in (12, 17) with 100 μg of His-Elongin A, 15 μg of His-Elongin B (17), and 15 μg of His-Elongin C (15). After dialysis, the renatured proteins were applied to a TSK SP-NPR column (35 mm by 4.6 mm, Hewlett-Packard) equilibrated in 40 mM Hepes-NaOH (pH 7.9), 0.1 mM EDTA, 1 mM DTT, 10% (v/v) glycerol, and 0.1 M KCI. The column was eluted at 0.6 ml/min at 8°C with a 9-ml gradient from 0.1 to 0.8 M KCI in the same buffer. Samples of each column fraction were analyzed by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and the proteins visualized by silver staining.
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33. Abbreviations for the amino acid residues are A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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37. We thank K. Garrett for the Elongin B and C clones and for support; D. Haque and Y. Takagi for help with purification of recombinant proteins; R. Robinson for peptide isolation; K. Payne for DNA sequencing; J. Minnerley and R. Wiegand for the rat brain cDNA library; P, Silverman, R. Klausner, A. Pause, R. Duan, G. Zurawski, S. Zurawski, and D, Liggett for helpful discussions; and K. Jackson for oligonucleotide synthesis. Supported by NIH grant GM41628 and by funds provided to the Oklahoma Medical Research Foundation by the H. A. and Mary K. Chapman Charitable Trust.