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A 30-kDa host protein binds to two very-late baculovirus promoters
1Eukaryotic Gene Expression Laboratory, National Institute of Immunology, New Delhi, India.
Abstract:
A 30-kDa host factor (polyhedrin-promoter-binding protein; PPBP) specifically binds to sequences critical for transcription from the baculovirus polyhedrin (p29) gene initiator promoter [Burma, S., Mukherjee, B., Jain, A., Habib, S. & Hasnain, S. E. (1994) J. Biol. Chem. 269, 2750-2757; Mukherjee, B., Burma, S. & Hasnain, S. E. (1995) J. Biol. Chem. 270, 4405-4411]. A host factor also binds, in gel shift assays, to the very-late p10 gene promoter through DNA sequence motifs similar to the PPBP.p29 interaction. The p10 host factor complex was specifically competed out with oligonucleotides containing p29 cognate sequence motifs AATAAA and TAAGTATT, but this did not occur when these motifs were replaced with random sequences. From ultraviolet cross-linking analysis, the molecular mass of this host factor was estimated to be approximately 30 kDa. Experiments were performed to investigate if this host factor displayed any differences in affinity and turnover with respect to the p29 and p10 untranslated leader sequences known to be important for temporal fine tuning and the late burst of transcription. Half-life determination of the p10-binding protein revealed similar binding affinities for the initiator elements of both the promoters, but higher affinity for the p10 5'-untranslated region (approximately 30 min versus approximately 10 min). The involvement of a similar host factor binding to both the p10 and p29 promoters indicates the possibility of a similar mode of transcription initiation from these two very-late promoters.
Insights
A 30-kDa host factor binds to baculovirus promoters, suggesting a shared mechanism for transcription initiation. This protein, polyhedrin-promoter-binding protein (PPBP), shows similar binding affinities for p29 and p10 promoters but higher affinity for the p10 5'-untranslated region.
Area of Science:
- Molecular Biology
- Virology
- Gene Regulation
Background:
- Baculovirus gene expression relies on specific host factors for transcription initiation.
- The polyhedrin (p29) and p10 genes are very-late promoters with distinct roles in the viral life cycle.
- Previous studies identified a 30-kDa host factor (PPBP) binding to the p29 promoter.
Purpose of the Study:
- To investigate the interaction of host factors with the very-late p10 baculovirus promoter.
- To determine if the host factor binding to the p10 promoter is similar to PPBP that binds to the p29 promoter.
- To compare the binding affinities and turnover rates of the host factor with p29 and p10 promoter elements.
Main Methods:
- Gel shift assays to detect protein-DNA interactions.
- Oligonucleotide competition assays to identify specific binding motifs.
- Ultraviolet cross-linking to estimate protein molecular mass.
- Half-life determination to assess binding kinetics.
Main Results:
- A 30-kDa host factor binds to the p10 promoter via DNA motifs similar to those recognized by PPBP.
- Binding to the p10 promoter was competed by p29 cognate sequences (AATAAA, TAAGTATT).
- The host factor exhibited similar binding affinities for the initiator elements of both p29 and p10 promoters.
- Higher binding affinity was observed for the p10 5 -untranslated region compared to the p29 promoter (approx. 30 min vs. 10 min half-life).
Conclusions:
- A similar 30-kDa host factor binds to both the very-late p29 and p10 baculovirus promoters.
- This suggests a conserved mechanism for transcription initiation from these promoters.
- The differential affinity for the 5 -untranslated regions may contribute to the temporal fine-tuning of gene expression.