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Molecular Endocrinology, Vol 8, 704-712, Copyright © 1994 by Endocrine Society

ARTICLES

Identification and characterization of the human transforming growth factor-alpha initiator

TH Shin and JE Kudlow
Department of Cell Biology, University of Alabama, Birmingham 35294.

Eukaryotic transcription requires promoter proximal elements. For class II promoters, two such elements are the TATA box and the initiator. The promoter for the human transforming growth factor-alpha (TGF alpha) gene has been shown to lack a TATA box, yet initiate transcription at a unique site. We have identified an approximately 13-basepair sequence between -5 and +8 as a new promoter element. We call this element the TGF alpha initiator based on the following observations: 1) it is located at the transcription initiation site; 2) the promoter activity is largely reduced by either deletion or mutation of the element; and 3) mutations result in initiation upstream of the authentic start site; the TGF alpha initiator directs site-specific initiation. An electrophoretic mobility shift assay demonstrated that a protein(s) in nuclear extracts forms complexes with the TGF alpha initiator. This interaction is sequence specific and depends on nucleotides that are critical for the promoter activity in vivo. Two polypeptides, 105 and 95 kilodaltons, were detected by Southwestern blot analysis on the basis that they specifically interact with the TGF alpha initiator. The larger polypeptide, TIBP-1, was subsequently purified by a matrix- immobilized TGF alpha initiator sequence and was shown to possess the TGF alpha initiator-specific mobility shift activity and an ability to interact with the initiator when immobilized on a membrane. In summary, we identified and characterized the TGF alpha initiator, a proximal element that is important for the accurate transcription of the TGF alpha gene, and the 105-kilodalton protein that interacts with this element.(ABSTRACT TRUNCATED AT 250 WORDS)


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