RNA Secondary Structure Folding Kinetics

Due to the ability to form enoumous secondary structures, RNAs can cover most aspects of biofunctions, including the gene conservation, replication, expression, regulation and so on. Therefore, aimming on the process of RNA sequence folding into its specific secondary functional structure, the folding kinetics becomes an important part in RNA research. The RNA folding kinetics, the cotranscription folding problem and the mechanism of riboswitches are studied in this thesis. The main research contents are as follows.1. Establish a computational approach of folding kinetics combining the free energy landscape and master equationThe vital part in RNA folding is forming the helix. Based on the free energy landscape, the growing of helix could be considered as a nucleation growth process: once the first several stacks are closed, the helix will form rapidly. Therefore we can identify the complete helix as the building block of secondary structures. Such clustering modeling method could greatly improve the computational efficiency, and made it possible to calculate the folding kenetics for large RNAs. In our approach, the basic kinetic step is forming/disrupting/exchanging a helix. We also provide the algorithm of calculating the rate constant of the three kinds of kinetic transition pathway. The testing result of our alogorithms with exact master equation method shows that our method is quite reliable. The helix exchanging pathway, which is termed the tunneling pathway because of its low barrier, would be significant in the structural rearrangement in RNA folding. Our computational results show good quantitative agreements with a nanomechanism RNA switch experiment, indicating that our method is quite reliable in predicting the structural transition pathway, the native structure and intermediate structures, the folding products and so on.2. Develop the computational approach to solve the RNA cotranscription folding problemOnce the nucleotide is newly synthesized by the RNA polymerase, the RNA chain elongates and brings different folding behavior, which is termed cotranscriptional folding. We develop the helix-based master equation method by modeling the transcription as step-wise process, in which each step is the duration of transcribe a nucleotide. For each step, the kinetics algorithm predicts the populational kinetics, transition pathways, folding intermediates and the folding products. By integrating the results of every step of transcription, we can predict the folding behavior of the whole transcription scenerio, which shows contrastingly different features than the renaturation folding. The competetion between the transcription speed and transition rate determines the transition pathway and the end product of folding. For example, fast transcription favors the formation of brach-like structures than rod-like structures, and cotranscription folding would reduce the probability of formation of misfolded structures. Our computation shows good agreement with the experimental data, suggests our method would be reliable for the quantitative prediction for RNA cotranscriptional folding.3. Study the dynamical control mechanism of riboswitches Riboswitch refers to some very important gene regulation system of non-coding RNAs. By binding with ligand molecule or not, the riboswitch molecule can form two distinctive structures, which would repress or activate the gene expression respectively. The riboswitch consist an aptamer platform and an overlapping expression platform. The aptamer platform can bind with ligand molecule and causes the structural rearrangement of the expression platform, which serves as on/off signals on translational and transcription level.The addA and pbuE riboswitches show distinctive folding behaviors:the full length pbuE riboswitch resist binding with the ligand after the transcription, while the full length addA fold into the ligand-binding complex structure. Our computation indicates that the aptamer platform of pbuE is just the intermediate structure during the transcription and would soon be displaced by the expression platform. The time window of aptamer platform is too short for the ligand to bind. However, the aptermer platform of addA riboswitch would occupy a high probability after the transcription, and the transition from aptamer platform to expression platform is difficult. Our computation can be helpful to understand the gene regulation mechanism of riboswitches.