Abstract
The human small nuclear (sn) RNA promoters contain a proximal sequence element (PSE), which recruits the basal transcription factor SNAPc, and a distal sequence element characterized by an octamer sequence, which recruits the POU domain transcription factor Oct-1. The Oct-1 POU domain and SNAPc bind cooperatively to probes containing a PSE and an octamer sequence, and this effect contributes to efficient transcription in vitro. In vivo, however, Oct-1 regions outside of the POU domain can activate snRNA gene transcription. Here, we have examined whether the role of these regions is to contribute to cooperative binding with SNAPc. We find that they indeed improve cooperative binding, but most of the effect is nevertheless mediated by just the POU domain. This suggests that Oct-1 activates transcription of snRNA genes in at least two steps, recruitment of SNAPc mediated primarily by the POU domain, and a later step mediated by regions outside of the POU domain. We also show that a PSE-binding complex observed in nuclear extracts consists of Oct-1 and SNAPc. Although Oct-1 cannot bind effectively to the PSE probe on its own, in the complex it contacts DNA. Thus, in a nuclear extract, SNAPc can recruit Oct-1 to a probe to which Oct-1 cannot bind on its own.
Highlights
Transcriptional activators are key regulators of RNA polymerase II transcription, but their mode of action is still poorly understood
We find that they do contribute to cooperative binding but most of the effect is mediated by the POU domain, suggesting that the Oct-1 activation domains play their primary role at a later step in the activation process
To determine (i) whether regions outside of the POU domain contribute to such recruitment of SNAPc to the proximal sequence element (PSE), and (ii) whether the Oct-1 regions outside of the POU domain are more active in this process than the Oct-2 regions outside of the POU domain, we compared the abilities of Oct-1, the Oct-1 POU domain (POU1), Oct-2, and the Oct-2 POU domain (POU-2) to recruit SNAPc to a PSE in an EMSA
Summary
Constructs for PCR Probes—The plasmids containing the H2B octamer site and human U6 PSE were previously described [15]. Probes were generated by PCR amplification of these constructs using the universal sequencing primer end-labeled with [g-32P]ATP and T4 polynucleotide kinase and the re-. Protein purity and concentration were assessed by the same method as with the POU domains above except that the protein gels were stained with silver For both Oct-1 and Oct-1.P.1, a number of truncated proteins were visible below the full-length products. The binding reactions were performed in a total volume of 20 ml containing final concentrations of 100 mM KCl, 20 mM HEPES, pH 7.9, 5 mM MgCl2, 0.2 mM EDTA, 10% glycerol, 20 mg of fetal calf serum as a protein carrier, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.4 mg each of poly(dI-dC) and pUC118. EMSA reactions containing 16 ml of SNAPc, O.4 mg of His-Oct-1, and 100,000 cpm of DNA probe in a total volume of 80 ml were performed as
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
