Investigation of the dynamics and conformational changes of the U1A protein

The U1A-SL2 RNA complex is a model system for studying interactions between RNA and the RNA recognition motif (RRM), which is one of the most common RNA binding domains. Despite extensive studies conducted to study the origins of U1A-RNA affinity and specificity, little is known about the kinetics and mechanism of binding. This dissertation aims to help understand the contributions of dynamic processes to the U1A-RNA complex formation. The first chapter introduces the significance of studying RNA-binding proteins, and RNA-Recognition Motif proteins in particular. It also presents the general dynamic processes that have been observed in RRMs. Chapter 2 is about the characterization of the dynamics of an essential helix in the U1A protein, helix C. The time-resolved anisotropy data obtained here supports the induced fit mechanism of binding. Chapter 3 describes the kinetic studies of dissociation of the U1A-SL2 RNA complex using laser T-jump and stopped-flow fluorescence methods. Analysis of the kinetic data suggested three phases of dissociation with two intermediate states. Chapter 4 is a follow up study of chapters 2 and 3 to further investigate helix C dynamics. The experimental design makes use of tryptophan fluorescence quenching with histidine and cysteine. Chapter 5 explores the existence of cooperative interactions in the U1A protein. Taken together, all these results begin to build a comprehensive picture of the complex dynamic processes involved in the formation of an RRM-RNA complex.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Anunciado, Divina B.

Contributors dc:contributor

• Baranger, Anne M.
• Gruebele, Martin
• van der Donk, Wilfred A.
• Zhao, Huimin

• conformational changes
• dynamics
• RNA-recognition motif
• cooperative interactions
• time-resolved fluorescence anisotropy
• temperature-jump kinetics
• stopped-flow kinetics

Statement dc:rights

• Copyright 2010 Divina B. Anunciado

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/18490

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/18490