Visualising RNA folding dynamics in crowded environments and with single-molecule resolution

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Title: Visualising RNA folding dynamics in crowded environments and with single-molecule resolution
Authors: Paudel, Bishnu
Item Type: Thesis or dissertation
Abstract: Cellular environments are highly crowded due to the presence of nucleic acids, proteins and other cellular organelles that can occupy between 20%- 40% of the total cellular volume. Biomolecules, such as RNAs, require precise folding to the most compact native state in order to perform their specific function inside the cell. How crowding environments can influence the folding, stability and functions of RNA molecules is largely unknown. Generally, RNA folding and catalysis are studied in dilute in vitro conditions, which do not truly mimic cellular environments. The work of this thesis combines single-molecule fluorescence resonance energy transfer (smFRET) and bulk cleavage assays to determine the effects of molecular crowding agents in folding and catalysis of model small RNA enzymes, specifically the hairpin ribozyme and the large group II intron ribozyme. The single molecule data show that PEG favours the formation of the most compact (active) structure of RNA enzymes by increasing the docking rate constants. In addition, ribozyme folding and catalysis occurs at low concentrations of Mg2+ in the presence of crowding agents. Lastly, ensemble radiolabeled cleavage assays have shown that molecular crowding agents accelerate ribozymes’ catalysis significantly at lower concentration of Mg2+. This study show that crowding agents influence the stability and the function of ribozymes. These results are consistent with the idea that in vivo RNA enzymes have evolved to function optimally within the crowded environment of the cell. In addition, to understand RNA-protein interactions, the function of Dbp2, a DEAD-box helicase at single molecule resolution has been characterised. The results show that Dbp2 can bind and open double stranded RNA without ATP. In the presence of ATP, the opening and closing events increases, indicating Dbp2 recycling occurs faster. Finally, I have observed that the activity of Dbp2 reduces in the presence of the protein, Yra1.
Content Version: Open Access
Issue Date: Jun-2015
Date Awarded: Aug-2015
URI: http://hdl.handle.net/10044/1/44275
Supervisor: Rueda, David
Sponsor/Funder: Imperial College London
Medical Research Council (Great Britain)
Funder's Grant Number: Imperial College startup fund and MRC (UK, MC_A658_5TY10)
Department: Department of Medicine
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses



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