Type of Document Dissertation Author Huang, Wei Author's Email Address firstname.lastname@example.org, email@example.com URN etd-10242011-135130 Title Structural and Mechanistic Studies on the Interactions between S-adenosylmethionine (SAM) and the SAM-I Riboswitch Degree Doctor of Philosophy (Ph.D.) Department Biochemistry (Biological Sciences) Advisory Committee
Advisor Name Title Aboul-ela, Fareed Committee Chair Lee, Yong-Hwan Committee Co-Chair Battista, John R. Committee Member Jha, Shantenu Committee Member Valverde, Rodrigo A. Committee Member Keywords
- conformational heterogeneity
- strand switching
- SAM-I riboswitch
- Molecular Dynamics simulation
- base pair probability
Date of Defense 2011-10-21 Availability restricted AbstractThe chemical and physical properties of RNAs create a diverse functional portfolio to influence the functional outcomes of genomes. One scheme is to recognize cognate small molecule metabolites and to adopt distinct conformations to adjust the gene expression. Segments of messenger RNAs (mRNAs) that adopt this scheme are called riboswitches. As potential targets for designing novel antibiotics and portable regulatory devices for synthetic biology, riboswitches have gained increasing attention. The key to understand the functionality encoded in a riboswitch sequence requires unveiling the mechanism of transmitting ligand recognition to gene expression.
In this work, both computational and experimental techniques are employed to investigate the link between cognate ligand binding and conformational rearrangement of the SAM-I riboswitch. This riboswitch modulates the biosynthetic pathways of methionine, cysteine, S-adenosylmethionine (SAM) and other sulfur containing metabolites at the transcriptional level. Molecular Dynamics (MD) simulation, with improved force field, extended time scale and empowered by advanced computer hardware, is used to explore the conformational dynamics in 3D space. A partition function approach is adopted to examine potential conformational heterogeneity within the functional decision windows of the SAM-I riboswitch during the synthesis of the transcript. A proposed framework combining RNA tertiary structure prediction with experimental observations and MD simulations appears to be plausible for modeling transient events during RNA folding. Finally, experimental techniques, such as chemical probing, equilibrium dialysis, UV melting, NMR spectroscopy and SAXS, are used to verify insights gained from computational work or to generate structural information for further computational structure modeling.
This work has the following implications: 1) SAM plays an important role in anchoring the junction between helices one and two (J1/2), facilitating formation of the "OFF" state conformer, which may be synergized with formation of a nearby Mg2+ binding site. 2) Alternative or "misfolded" conformations due to the interactions between J1/2 and decoy regions representing the "ON" state ensemble facilitate fine tuning of the SAM-I riboswitches. 3) Simulated strand switching within hybrid intermediate structures in the presence of SAM reveals atomic level details of SAM-induced stabilization of the transcriptional OFF state.
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