title: Monomer and dimer of Chandipura virus unphosphorylated P-protein binds leader RNA differently: implications for viral RNA synthesis.
abstract: Interaction of the leader RNA with the unphosphorylated P-protein has been proposed to play a key role in the transcription-replication transition of Chandipura virus, a model rhabdovirus. Electrophoretic mobility shift assay with the leader RNA and the unphosphorylated P-protein demonstrated existence of two distinct complexes in vitro. Measurements of stoichiometry indicate the protein monomer/RNA ratio to be 1:1 and 2:1 for faster and slower migrating bands, respectively. We have also observed a concentration-dependent oligomerization of the unphosphorylated P-protein, in sub-micromolar to low micromolar range. Sedimentation velocity, dynamic light scattering and large zone gel filtration experiments suggest a monomer-dimer-tetramer model of association. RNA binding experiments suggest that the two complexes assembled from one molecule of the leader RNA binding to either a protein monomer or a dimer. A truncated RNA consisting of a 3' region of the leader transcript exclusively formed the 1:1 complex, whereas a RNA consisting of only the 5' region forms the 2:1 complex exclusively. RNA binding experiments at different protein concentrations suggest that binding of the RNA comprising the 3' region weakens significantly at higher P(0) concentrations, whereas in contrast the binding of the RNA comprising the 5' region becomes modestly tighter. Implications of two different types of leader RNA-P-protein complexes in viral RNA synthesis are discussed.
authors.affiliation: Department of Biochemistry and Dr B. C. Guha Centre for Genetic Engineering and Biotechnology, University College of Science, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700 019, India.
authors.forename: Soumen
authors.initials: S
authors.lastname: Basak
authors.forename: Smarajit
authors.initials: S
authors.lastname: Polley
authors.forename: Mausumi
authors.initials: M
authors.lastname: Basu
authors.forename: Dhrubajyoti
authors.initials: D
authors.lastname: Chattopadhyay
authors.forename: Siddhartha
authors.initials: S
authors.lastname: Roy
mesh_terms: D046911:Macromolecular Substances; D018832:Molecular Chaperones; D009690:Nucleic Acid Conformation; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D012367:RNA, Viral / Q000378:metabolism*; D014158:Transcription, Genetic; D014364:Tryptophan / Q000737:chemistry; D018116:Vesiculovirus / Q000378:metabolism; D015678:Viral Structural Proteins / Q000737:chemistry* / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D046911:Macromolecular Substances; D018832:Molecular Chaperones; C412837:P protein, Chandipura virus; D010750:Phosphoproteins; D012367:RNA, Viral; D015678:Viral Structural Proteins; D014364:Tryptophan