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Transcriptional analysis of minute virus of mice P4 promoter mutants
1Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.
Abstract:
A series of 5' deletion, internal deletion, and linker-scanning mutants of the minute virus of mice P4 promoter were constructed and analyzed for transcriptional activity in nuclear extracts of mouse A92L fibroblasts. A GC box and a TATA box essential for in vitro transcription from the P4 promoter were localized between nucleotides 150 and 180 (-55 to -25 relative to the primary RNA start site). Although this region also exhibited homologies to other transcriptional control elements, the simian virus 40 enhancer, and the adenovirus E1A enhancer, only the GC box and TATA box appear functional. These two motifs also play an essential role in vivo, although additional upstream sequences (between -139 and -55) are required for optimal transcription. DNase I footprinting, competitive gel retardation assays, and UV-photocrosslinking were used to identify Sp1-like proteins of 95 and 120 kilodaltons in A92L extracts that interact with the GC box of the minute virus of mice P4 promoter.
Insights
Researchers identified essential GC and TATA boxes for Minute Virus of Mice P4 promoter transcription. Sp1-like proteins bind to the GC box, crucial for gene expression in vitro and in vivo.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- The Minute Virus of Mice (MVM) P4 promoter regulates viral gene expression.
- Understanding promoter function is crucial for viral replication and gene regulation studies.
Purpose of the Study:
- To delineate the functional elements of the MVM P4 promoter.
- To identify proteins interacting with the MVM P4 promoter.
Main Methods:
- Construction and analysis of MVM P4 promoter mutants (deletion, linker-scanning).
- In vitro transcription assays using mouse fibroblast nuclear extracts.
- DNase I footprinting, gel retardation, and UV-photocrosslinking assays.
Main Results:
- A GC box and a TATA box (nucleotides -55 to -25) are essential for in vitro P4 promoter transcription.
- These motifs are also critical in vivo, with upstream sequences (-139 to -55) enhancing transcription.
- Sp1-like proteins (95 and 120 kDa) were identified that bind to the P4 promoter's GC box.
Conclusions:
- The GC box and TATA box are key functional elements of the MVM P4 promoter.
- Specific Sp1-like proteins interact with the GC box, mediating transcriptional activity.
- Optimal P4 promoter function requires both core elements and upstream regulatory sequences.