A Tale of Two RNAs: Single Molecule Investigation of the Conformation, Dynamics and Ligand Binding to the PreQ1 and T-box Riboswitches.
dc.contributor.author | Suddala, Krishna Chaitanya | en_US |
dc.date.accessioned | 2014-10-13T18:20:01Z | |
dc.date.available | NO_RESTRICTION | en_US |
dc.date.available | 2014-10-13T18:20:01Z | |
dc.date.issued | 2014 | en_US |
dc.date.submitted | 2014 | en_US |
dc.identifier.uri | https://hdl.handle.net/2027.42/108919 | |
dc.description.abstract | Riboswitches are structured mRNA domains that can bind cellular metabolites and control gene expression of downstream genes mainly via transcription attenuation or inhibition of translation initiation. Although structures of many ligand-bound riboswitches are available, knowledge on their ligand-free conformations is scarce. Subsequently, the ligand-mediated folding process of riboswitches is poorly understood. In this dissertation, we used single molecule FRET to investigate the conformation and ligand binding properties of two very distinct riboswitches. We showed that, contrary to previous studies, the structurally similar but functionally different preQ1 riboswitches from B. subtilis (Bsu) and T. tencongensis (Tte) have similar conformational ensemble in their ligand-free state with only subtle differences in their dynamics. Our smFRET data in combination with computational simulations suggested that both the riboswitches adopt ligand-free ‘pre-folded’ conformations and fold through distinct pathways that are similar to the conformational selection and induced fit mechanisms, respectively. We also demonstrated how remote mutations can affect the ligand binding affinities of riboswitches. Later, using smFRET, we probe the effect of various ligands on the kinetics of the Bsu riboswitch conformational dynamics with an aim to dissect its ligand binding mechanism. Our data suggest that the Bsu riboswitch can fold through both induced fit or conformational selection pathways, the relative extent of which is dependent on the presence of Mg2+. The T-box riboswitch is one of the complex riboswitches that binds tRNA and controls gene expression by sensing the relative levels of charged and uncharged tRNA. The structure of a T-box riboswitch stem-I:tRNA complex was recently solved, but it lacks the important genetic regulatory domain. By using various designs of the glyQS T-box riboswitch, we have studied the global conformation of the full T-box riboswitch and estimated distances between different regions. We measured tRNA binding kinetics to different T-box variants and showed that the double T-loop motif only contributes modestly to decrease the tRNA dissociation rate. Further, we directly demonstrated that the presence of glycine increases the tRNA dissociation rate ~6-fold that forms the basis of T-box riboswitch mechanism. Based on our kinetic data, we propose an improved kinetic model of the T-box riboswitch function. | en_US |
dc.language.iso | en_US | en_US |
dc.subject | Riboswitches | en_US |
dc.subject | RNA Structure and Dynamics | en_US |
dc.subject | Single Molecule FRET | en_US |
dc.subject | RNA Folding | en_US |
dc.subject | Ligand Binding | en_US |
dc.subject | Binding Kinetics | en_US |
dc.title | A Tale of Two RNAs: Single Molecule Investigation of the Conformation, Dynamics and Ligand Binding to the PreQ1 and T-box Riboswitches. | en_US |
dc.type | Thesis | en_US |
dc.description.thesisdegreename | PhD | en_US |
dc.description.thesisdegreediscipline | Biophysics | en_US |
dc.description.thesisdegreegrantor | University of Michigan, Horace H. Rackham School of Graduate Studies | en_US |
dc.contributor.committeemember | Walter, Nils G. | en_US |
dc.contributor.committeemember | Cheng, Wei | en_US |
dc.contributor.committeemember | Saper, Mark A. | en_US |
dc.contributor.committeemember | Brooks Iii, Charles L. | en_US |
dc.contributor.committeemember | Al-Hashimi, Hashim M. | en_US |
dc.subject.hlbsecondlevel | Biological Chemistry | en_US |
dc.subject.hlbsecondlevel | Chemistry | en_US |
dc.subject.hlbsecondlevel | Physics | en_US |
dc.subject.hlbtoplevel | Science | en_US |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/108919/1/krishnac_1.pdf | |
dc.owningcollname | Dissertations and Theses (Ph.D. and Master's) |
Files in this item
Remediation of Harmful Language
The University of Michigan Library aims to describe library materials in a way that respects the people and communities who create, use, and are represented in our collections. Report harmful or offensive language in catalog records, finding aids, or elsewhere in our collections anonymously through our metadata feedback form. More information at Remediation of Harmful Language.
Accessibility
If you are unable to use this file in its current format, please select the Contact Us link and we can modify it to make it more accessible to you.