ABSTRACT

We have used an in vitro RNA polymerase II (RNAP II) inhibition-restimulation assay to investigate the inability of a form of RNAP II (RNAP IIB) that lacks the conserved, C-terminal heptapeptide repeat domain (CTD) to transcribe the dihydrofolate reductase (dhfr) promoter. Our previous studies demonstrated promoter-specific responses to RNAP IIB in the inhibition-restimulation assay and suggested the existence of cis-acting elements that alleviate the requirement for the CTD. We have now identified elements from two different classes of promoters that can convert dhfr to a CTD-independent promoter. First, addition of a consensus TATA box to the dhfr promoter resulted in a promoter capable of CTD-independent transcription and increased the promoter's affinity for the general transcription factor TFIID. These results suggest that high affinity for TFIID correlates with an ability to be transcribed by RNAP IIB, supporting a proposed interaction between the CTD and TFIID. Second, transfer of a combination of two elements (located at -25 and +1) from the rep-3b promoter, which does not contain a consensus TATA box but can nonetheless be transcribed by RNAP IIB, into the dhfr promoter also allowed CTD-independent transcription. These elements do not constitute a high affinity binding site for TFIID, indicating that an additional mechanism exists to allow CTD-independent transcription. Thus, elements from two classes of CTD-independent promoters that can obviate a requirement for the CTD appear to function via distinct mechanisms. Our finding that a change in a basal element can affect a requirement for the CTD is consistent with a role for the CTD during the formation of the transcriptional preinitiation complex.

FOOTNOTES

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This work was supported by United States Public Health Service Grants CA45240 and CA59524 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported by United States Public Health Service Training Grant GM07215 from the National Institutes of Health.

¶

To whom correspondence should be addressed: McArdle Laboratory, 1400 University Ave., Madison, WI 53706. Tel.: 608-262-2071; Fax: 608-262-2824.

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The abbreviations used are: CTD, C-terminal heptapeptide repeat domain; RNAP, RNA polymerase; mAb, monoclonal antibody; CAD, carbamoyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase; GMSA, gel mobility shift assay; TF, transcription factor; TBP, TATA-binding protein; TAF, TBP-associated factor; HIP, housekeeping initiation protein; Irf, interferon regulatory factor; bp, base pair(s).

()

L. A. Strasheim and R. R. Burgess, unpublished data.

()

N. E. Thompson and R. R. Burgess, unpublished data.

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