Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have identified a component of the eukaryotic RNA polymerase II transcriptional machinery that is more heat-labile than TFIID. DHFR transcriptional activity was severely reduced in 40 degrees C heat-treated extracts in which TFIID was fully active. This heat-labile activity was required for the transcription of both TATA box and non-TATA box promoters that are activated by the transcription factor Sp1. Gel mobility shifts indicated that Sp1 DNA binding activity was heat-labile, and the addition of purified Sp1 to 40 degrees C heat-treated extracts fully restored DHFR transcriptional activity. In contrast, the addition of Sp1 to 47 degrees C heat-treated extract did not result in transcriptional activity from the DHFR promoter. We conclude that reduction in Sp1 DNA binding activity is partially responsible for the heat-sensitive loss of DHFR transcriptional activity, but that a second essential activity is also inactivated by 47 degrees C heat-treatment. The discovery of this heat-labile component of Sp1 activation has two important implications in the analysis of transcriptional regulation. First, it demonstrates that heat-treated extracts are not appropriate for examination of the involvement of TFIID in the transcription of Sp1-activated promoters. Second, it explains the previously reported low-temperature optima for transcription from the DHFR promoter and demonstrates that transcriptional studies of Sp1-activated promoters should not be performed at 30 degrees C.
 ...
PMID:Sp1 activation of RNA polymerase II transcription complexes involves a heat-labile DNA-binding component. 182 Feb 11

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.
 ...
PMID:Identification of cis-acting elements that can obviate a requirement for the C-terminal domain of RNA polymerase II. 789 26