http://192.168.1.40:8080/xmlui/handle/123456789/2443 2026-05-31T14:24:49Z 2026-05-31T14:24:49Z Roy, Siddhartha http://192.168.1.40:8080/xmlui/handle/123456789/2453 2013-04-02T12:49:00Z 2004-01-01T00:00:00Z

Fluorescence quenching methods to study protein-nucleic acid interactions Roy, Siddhartha

2004-01-01T00:00:00Z Basak, S. Polley, S. Basu, M. Chattopadhyay, D. Roy, Siddhartha http://192.168.1.40:8080/xmlui/handle/123456789/2448 2013-04-02T12:27:26Z 2004-06-18T00:00:00Z

Monomer and dimer of Chandipura virus unphosphorylated P-protein binds leader RNA differently: Implications for viral RNA synthesis Basak, S.; Polley, S.; Basu, M.; Chattopadhyay, D.; Roy, Siddhartha 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 I : I 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, 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. DOI: 10.1016/j.jmb.2004.03.081

2004-06-18T00:00:00Z Roy, Siddhartha Semsey, S. Liu, M. Gussin, G. N. Adhya, S. http://192.168.1.40:8080/xmlui/handle/123456789/2444 2013-04-02T12:05:28Z 2004-11-26T00:00:00Z

GaIR represses gaIP1 by inhibiting the rate-determining open complex formation through RNA polymerase contact: A GaIR negative control mutant Roy, Siddhartha; Semsey, S.; Liu, M.; Gussin, G. N.; Adhya, S. Ga1R represses the galP1 promoter by a DNA looping-independent mechanism. Equilibrium binding of Ga1R and RNA polymerase to DNA, and real-time kinetics of base-pair distortion (isomerization) showed that the equilibrium dissociation constant of RNA polymerase-P1 closed complexes is largely unaffected in the presence of saturating Ga1R, indicating that mutual antagonism (steric hindrance) of the regulator and the RNA polymerase does not occur at this promoter. In fluorescence kinetics with 2-AP labeled P1 DNA, Ga1R inhibited the slower of the two-step base-pair distortion process. We isolated a negative control Ga1R mutant, S29R, which while bound to the operator DNA was incapable of repression of P1. Based on these results and previous demonstration that repression requires the C-terminal domain of the alpha subunit (alpha-CTD) of RNA polymerase, we propose that Ga1R establishes contact with alpha-CTD at the last resolved isomerization. intermediate, forming a kinetic trap. DOI: 10.1016/j.jmb.2004.09.070

2004-11-26T00:00:00Z