The 5′‐untranslated RNA of the human dhfr minor transcript alters transcription pre‐initiation complex assembly at the major (core) promoter

Abstract

The human dhfr minor transcript is distinguished from the predominant dhfr mRNA by an ∼400 nucleotide extension of the 5′‐untranslated region, which corresponds to the major (core) promoter DNA (its template). Based on its unusual sequence composition, we hypothesized that the minor transcript 5′‐UTR might be capable of altering transcription pre‐initiation complex assembly at the core promoter, through direct interactions of the RNA with specific regulatory polypeptides or the promoter DNA itself. We found that the minor transcript 5′‐UTR selectively sequesters transcription factor Sp3, and to a lesser extent Sp1, preventing their binding to the dhfr core promoter. This allows a third putative transcriptional regulatory protein, which is relatively resistant to sequestration by the minor transcript RNA, the opportunity to bind the dhfr core promoter. The selective sequestration of Sp3 > Sp1 by the minor transcript 5′‐UTR involves an altered conformation of the RNA, and a structural domain of the protein distinct from that required for binding to DNA. As a consequence, the minor transcript 5′‐UTR inhibits transcription from the core promoter in vitro (in trans) in a concentration‐dependent manner. These results suggest that the dhfr minor transcript may function in vivo (in cis) to regulate the transcriptional activity of the major (core) promoter.