Purification of human transcription factor IIIA and its interaction with a chemically synthesized gene encoding human 5 S rRNA.
Transcription factor IIIA (TFIIIA) was purified from cytoplasmic extracts of HeLa cells by developing a simple and efficient procedure employing phosphocellulose under widely differing ionic conditions followed by affinity chromatography on immobilized human 5 S genes. This procedure yielded a fraction containing human TFIIIA activity and a protein of 35 kDa as its major component. Moreover, we succeeded in renaturing the activity of human transcription factor IIIA (hTFIIIA) isolated after preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in identifying a polypeptide of 35 kDa with the transcription activity. This value differs from that reported for Xenopus TFIIIA. It could be demonstrated by footprinting analyses that hTFIIIA specifically binds to the internal control region of the human 5 S rRNA gene. The limits of protection slightly differ at the 3' border of the internal control region from those imprinted by Xenopus TFIIIA on the same gene. Comparative footprint analyses of hTFIIIA on the human and frog somatic 5 S rRNA gene, measured in titration, competition, and salt-stability experiments, demonstrated a higher affinity of the human factor to the homologous gene. These results, together with the difference in molecular mass of these functionally analogous proteins, reemphasize the importance of homologous systems for the analysis of mechanisms involved in gene regulation.
Seifart
K H
KH
Institut für Molekularbiologie and Tumorforschung, Marburg/Lahnberge, Federal Republic of Germany.
Wang
L
L
Waldschmidt
R
R
Jahn
D
D
Wingender
E
E
D004591:Electrophoresis, Polyacrylamide Gel; D005796:Genes; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D008722:Methods; D008970:Molecular Weight; D011489:Protein Denaturation; D012335:RNA, Ribosomal / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000378:metabolism*; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000302:isolation & purification*
D012335:RNA, Ribosomal; D012341:RNA, Ribosomal, 5S; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors