Identification of cis-acting elements that can obviate a requirement for the C-terminal domain of RNA polymerase II.
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.
Buermeyer
A B
AB
McArdle Laboratory for Cancer Research, Medical School, University of Wisconsin, Madison 53706.
Strasheim
L A
LA
McMahon
S L
SL
Farnham
P J
PJ
D001483:Base Sequence; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000737:chemistry / Q000502:physiology*; D016385:TATA Box / Q000502:physiology*; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic
D035362:Transcription Factor TFIID; D014157:Transcription Factors; D012319:RNA Polymerase II