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DNA binding activity of the baculovirus late expression factor PP31
Linda A Guarino1, Toni-Ann Mistretta, Wen Dong
1Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA. lguarino@tamu.edu
Virus Research
|November 30, 2002
Summary
The baculovirus PP31 protein, essential for viral gene expression, binds DNA and inhibits transcription. This suggests other factors are needed to help viral RNA polymerase overcome PP31 binding during late gene expression.
Area of Science:
- Molecular Biology
- Virology
- Gene Expression
Background:
- PP31 is a crucial baculovirus protein for viral late gene expression.
- Understanding PP31's role in transcription is key to deciphering viral replication mechanisms.
Purpose of the Study:
- To investigate the function of PP31 in baculovirus late transcription in vitro.
- To characterize the DNA-binding properties and transcriptional effects of purified PP31.
Main Methods:
- Purification of native, non-tagged PP31 protein from infected insect cells using sequential chromatography (heparin affinity, cation exchange, gel filtration).
- DNA binding assays to determine PP31's affinity for single- and double-stranded DNA.
- In vitro transcription assays using purified baculovirus RNA polymerase.
Main Results:
- PP31 was purified in large quantities (approx. 5 mg), confirming its abundance.
- PP31 demonstrated equal binding affinity for both single- and double-stranded DNA.
- PP31 addition strongly inhibited in vitro transcription by baculovirus RNA polymerase.
Conclusions:
- PP31 binds to DNA and acts as a potent inhibitor of baculovirus transcription.
- The inability of RNA polymerase to displace PP31 suggests a requirement for additional late expression factors to facilitate transcription initiation on PP31-coated DNA templates.