[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The Ribosome is the compact nanomachine with multiple coordinated components working together to translate the genetic code in the mRNA into linear protein sequence. Since it was discovered in 1955 by George Emil Palade, the research around ribosome has been pushed forward by hundreds of labs worldwide. In 2000, the ribosome structures at atomic resolution were resolved, which open a new era for ribosome study. Structures help people design and explain the biochemical data deeper and better. However, ribosome is not static and it is a dynamic and highly regulated machine. The function of ribosome can be understood further only if we can follow the dynamics of individual components in different functional states. Solution NMR is a powerful technique for studying protein dynamics. However the gigantic nature of ribosome makes this task daunting. Thanks to the development of single molecule techniques, ribosome tRNA translocation and intersubunit rotation have been studied and produced new information about ribosome function Both single molecule FRET and optical tweezers have been successfully used to address the dynamic process of protein translation. In 2008, Dr. Peter Cornish and Dr. Dmitri Ermolenko followed the ribosome intersubunit rotation and L1 stalk dynamics in real time during the process of translocation, which was the first direct evidence of ribosome dynamics itself since the previous study inferred the ribosome dynamics from tRNA period. The previous study could not exclude the possibility that the observed dynamics resulted purely from tRNA. Cornish and Ermolenko concluded that ribosome dynamics is a spontaneous process that is driven by thermodynamic Brownian motion. This pioneering study open a window to address many unresolved problems such as the perturbation of dynamic effect by structured RNA, ribosome unwinding, and frameshifting. We found that the presence of RNA structure induces the ribosome into a new FRET state that we named it super rotated state. The population distribution of the super rotated state is correlated with the thermostability and the distance of RNA structure to the ribosome. Using other RNA structures like DNA:RNA hybrid and pseudoknot, the ribosome can also be induced into the super rotated state. Structured RNA inhibits the regular intersubunit rotation and drives the ribosome into the super rotated state. However, the structured RNA cannot stop the opento-close transition of the L1 stalk, which still can fluctuate between three different functional states. These results propose that the ribosome dynamics is composed of several independent units with their own identity. Since the super rotated state also can be induced by DNA:RNA hybrid, we can investigate how far when the RNA structure is away from the ribosome that the ribosome can sense the presence of RNA structures. When the DNA:RNA is 1-2 nucleotide away from the ribosome mRNA entrance tunnel, the intersubunit rotation still can fluctuate among three states and induce the hyper rotated state. We also studied the correlation between thermal stability and the percentage of hyper rotated state. The higher thermal stability indicates a higher percentage of hyper rotated state. The hyper rotated state is not RNA structure specific as long as the RNA structure is stably enough, which will create problem for ribosome to unwind. It is possible whenever ribosome cannot unwind the downstreat RNA structure is a specific time window such as the time for ribosome to read one genetic codon (~2s), ribosome's intersubunit rotation dynamics is out of balance and stay in a trapped hyper rotated state as long as the barrier is to not strong enough to hold it more than 2 seconds. The ribosome unwinding also can be observed when there is no additional factors present, which also confirms that ribosome itself is a helicase. Ribosome dynamics is the main theme of this dissertation. However, since I have worked on NMR dynamics for two years with Dr. Chun Tang, it is also an integral part of my technical Ph.D training. There are two proteins I worked on: mouse adiponectin, glutamine binding protein (QBP) and EIN-Hpr complex. The last three chapters include the summary of our study on these three proteins. We have completed backbone assignments for both adipotectin and QBP. For adiponectin, we characterized the ligand binding property by chemical shift perturbation and measured the calcium binding affinity with terbium luminescence resonance energy transfer. For the QBP, we designed a linker at the back of the glutamine binding pocket so we can control the magnitude of opening between two lobes of the binding site. We measured the glutamine binding affinity for different mutants and found the correlation with the ligand binding affinity and the magnitude of opening. In addition, we developed a new PRE method called differentially scaled PRE.
Influence of Downstream DNA/RNA Structure on Intersubunit Rotation of the Ribosome
