Poster abstracts
Poster number 15 submitted by Max Gilliland
Using Molecular Dynamics to Characterize Mutants in the Connector Region of Rho
Max Gilliland, Nicholas Sunday, Marcos Sotomayor, Irina Artsimovich
Abstract:
Transcription termination factors are necessary to silence synthesis of aberrant RNAs. Bacterial Rho protein is an archetype of factor-dependent termination. In Escherichia coli, Rho inhibits expression of anti-sense, corrupted, and horizontally-acquired RNA messages. E. coli Rho is a hexamer made up of 419 amino acids per subunit. Rho is an ATP-dependent, RecA-family hexameric helicase composed of an N-terminal RNA-binding and a C-terminal ATPase/helicase domains separated by a flexible 30-residue long connector region. A key step in the Rho mechanism is a switch from an open, RNA-loading state into a closed, translocation-competent state in which the RNA is captured inside the inner pore; this switch is activated by Rho binding to canonical RNA sequences yet Rho also has to act on non-canonical sites. Our genetic data suggest that the flexible connector region may modulate the transition between Rho's inactive open and active closed states. We have identified substitutions that confer defects in Rho-dependent termination and are predicted to reduce connector flexibility (e.g., Gly→Asp). We hypothesize that the connector region is involved allosterically in binding to of divergent RNA sequences and is a potential target for factors that control Rho activity. Using molecular dynamic simulation software NAMD, we can develop a model of the connector region by mutating residues in silico. By modeling the closure of Rho mutants, we can assess the contributions of individual amino acid residues to Rho function. Quantitative analysis of root-mean-square deviation of atomic positions and inner pore diameter along with viewing the mechanism with VMD modeling software can give insights to how the selected substitutions affect ring closure. Additionally, elucidating the molecular details of Rho action will lead to understanding of other motor proteins that couple ATP hydrolysis to translocation on polymer substrates.
Keywords: Transcription, Termination Factor, Molecular Dynamics