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A single-stranded promoter for RNA polymerase III.
Oliver Schroder1, E Peter Geiduschek, George A Kassavetis
1Division of Biological Sciences and Center for Molecular Genetics, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0634, USA.
Summary
Single DNA strands can initiate transcription. Researchers discovered that the nontranscribed strand of the Saccharomyces cerevisiae U6 snRNA gene acts as a promoter, directing RNA polymerase III transcription.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Promoters typically involve double-stranded DNA.
- Single-stranded DNA promoters are rare and poorly understood.
- The Saccharomyces cerevisiae U6 snRNA gene's promoter function was previously uncharacterized.
Purpose of the Study:
- To investigate the promoter capacity of individual DNA strands.
- To determine if the nontranscribed strand of the U6 snRNA gene can direct transcription.
- To characterize the elements and mechanism of this novel promoter.
Main Methods:
- In vitro transcription assays using RNA polymerase III.
- Site-directed mutagenesis to identify essential promoter elements.
- Analysis of transcription initiation factor IIIB (TFIllB) requirements.
Main Results:
- The nontranscribed strand of the U6 snRNA gene functions as a promoter.
- This promoter requires transcription initiation factor IIIB (TFIllB) for accurate initiation.
- The nontranscribed strand promoter is extensive, including the TATA box, a T(7) tract, and an upstream region.
- The template strand's 3' end must be within the transcription bubble, suggesting a role in RNA polymerase recruitment but not promoter opening.
Conclusions:
- A novel single-stranded DNA promoter mechanism is described for the U6 snRNA gene.
- The nontranscribed strand acts as a scaffold for RNA polymerase recruitment.
- The nontranscribed strand promoter is deficient in facilitating promoter opening by TFIllB.