The 30-kDa protein binding to the "initiator" of the baculovirus polyhedrin promoter also binds specifically to the coding strand.
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.
Mukherjee
B
B
Eukaryotic Gene Expression Laboratory, National Institute of Immunology, New Delhi, India.
Burma
S
S
Hasnain
S E
SE
D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019476:Insect Proteins; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019476:Insect Proteins; D009687:Nuclear Proteins; C085605:polyhedrin promoter-binding protein, insect