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Nucleic Acids Research, 2000, Vol. 28, No. 9 1986-1993
© 2000 Oxford University Press

Identification of a transcription factor IIIA-interacting protein

Rodney J. Moreland1,2, Michael E. Dresser2, Justin S. Rodgers1, Bruce A. Roe3, Joan W. Conaway2,4, Ronald C. Conaway2 and Jay S. Hanas1,*

1Department of Biochemistry and Molecular Biology, University of Oklahoma College of Medicine, Oklahoma City, OK 73104, USA, 2Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA, 3Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA and 4Howard Hughes Medical Institute, Oklahoma City, OK 73104, USA

Transcription factor IIIA (TFIIIA) activates 5S ribosomal RNA gene transcription in eukaryotes. The protein from vertebrates has nine contiguous Cys2His2 zinc fingers which function in nucleic acid binding, and a C-terminal region involved in transcription activation. In order to identify protein partners for TFIIIA, yeast two-hybrid screens were performed using the C-terminal region of Xenopus TFIIIA as an attractor and a rat cDNA library as a source of potential partners. A cDNA clone was identified which produced a protein in yeast that interacted with Xenopus TFIIIA but not with yeast TFIIIA. This rat clone was sequenced and the primary structure of the human homolog (termed TFIIIA-intP for TFIIIA-interacting protein) was determined from expressed sequence tags. In vitro interaction of recombinant human TFIIIA-intP with recombinant Xenopus TFIIIA was demonstrated by immuno­precipitation of the complex using anti-TFIIIA-intP antibody. Interaction of rat TFIIIA with rat TFIIIA-intP was indicated by co-chromatography of the two proteins on DEAE-5PW following fractionation of a rat liver extract on cation, anion and gel filtration resins. In a HeLa cell nuclear extract, recombinant TFIIIA-intP was able to stimulate TFIIIA-dependent transcription of the Xenopus 5S ribosomal RNA gene but not TFIIIA-independent transcription of the human adenovirus VA RNA gene.

* To whom correspondence should be addressed. Tel: +1 405 271 2995; Fax: +1 405 271 5440; Email: jay-hanas@ouhsc.edu


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