Abstract
We previously reported the purification and characterization of the polyhedrin promoter-binding protein (PPBP), an unusual DNA-binding protein that interacts with transcriptionally important motifs of the baculovirus polyhedrin gene promoter (S. Burma, B. Mukherjee, A. Jain, S. Habib, and S.E. Hasnain, J. Biol. Chem. (1994) 269, 2750-2757. PPBP also exhibits a sequence-specific single-stranded DNA-binding activity. Gel retardations and competition analyses with double- and single-stranded oligonucleotides indicated that PPBP binds the coding strand and not the noncoding strand of the promoter. This was further confirmed by UV cross-linking and Southwestern blotting experiments. Gel retardations with mutated oligonucleotides indicated that both dsDNA and ssDNA binding involve common AATA-AATAAGTATT motifs. However, ssDNA binding is dependent upon ionic interactions unlike dsDNA binding, which is mainly through nonionic interactions. The affinity of PPBP for the coding strand appears to be higher than that for duplex promoter DNA. Interestingly, the PPBP-coding strand complex has a longer half-life (approximately 60 min) than the PPBP-duplex promoter complex (approximately 15 min). PPBP represents a unique example of an "initiator" promoter-binding protein with dual dsDNA and ssDNA binding activities, and this reconciles very well with the unusual binding characteristics displayed by it. The formation of the PPBP-coding strand complex in vivo may be a crucial step for the exceptionally high and repeated rounds of transcriptional activity of the baculovirus polyhedrin gene promoter.
Highlights
From the Eukaryotic Gene Expression Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India
We previously reported the purification and characterization of the polyhedrin promoter-binding protein (PPBP), an unusual DNA-binding protein that interacts with transcriptionally important motifs of the baculovirus polyhedrin gene promoter
The baculovirus expression vector system is widely used for heterologous gene expression, little is known about the regulation of the polyhedrin promoter and the mechanism responsible for hypertranscription from this promoter
Summary
Gel retardations and competition analyses with double- and singlestranded oligonucleotides indicated that PPBP binds the coding strand and not the noncoding strand of the promoter. This was further confirmed by UV crosslinking and Southwestern blotting experiments. The formation ofthe PPBP-coding strand complex in vivo may be a crucial step for the exceptionally high and repeated rounds of transcriptional activity of the baculovirus polyhedrin gene promoter. In the baculovirus expression vector system, the very late polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus is used to direct the expression of foreign genes (Luckow, 1991; Jarvis and Summers, 1992; O'Reilly et al, 1992). B. acknowledge the award of research fellowships from the Council for Scientific and Industrial Research, Government of India
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