Abstract
摘要:本跨領域計畫長程目標為利用嶄新之單分子(single-molecule)分析技術並配合生化、生物物理與計算生物學的工具,探究核醣體各個組成份子於蛋白質轉譯作用(protein translation)中所扮演之功能與機制。在這個三年期計畫中,則將針對訊息核醣核酸上的二級結構在轉譯過程中的解旋與折疊對核醣體的解旋功能以及-1 轉譯框架移轉造成的影響,進行深入之探討,並期藉此釐清核醣體解旋功能的運作機制。子計畫一著重在運用生化方法來追蹤可以影響-1 轉譯框架移轉的核醣核酸結構之解旋與折疊。包括藉由核醣核酸2’hydroxyl group 的化學反應性追蹤與分析核醣核酸結構於轉譯過程中之解旋與折疊現像,以及以紫外光交叉連結(UV-crosslinking)界定-1 轉譯框架移轉減弱子與核醣體之交互作用。子計畫二將以單分子技術研究RNA 結構在有無核醣體存在時的變化。光鉗將用來測量刺激子hTPK-DU177 及其突變體的結構穩定度是否和其誘導框架位移的能力有關。同時,在核醣體作用下,光鉗和單分子FRET 也將運用來分析刺激子及減弱子結構的動力學特性。子計畫三則將運用各種液態核磁共振光譜學技術,諸如磁共振氫氘置換和原子核秩序參數的量測來進行核酸結構動態與摺疊分析,並探討二級結構變構(allostery)及摺疊合作性(foldingcooperativity)。子計畫四將以電腦模擬及適切的物理模型來解析核醣體的機械性質並且找出-1 框架移轉(-1 PRF)可能的異構調控機制。現有大腸桿菌核醣體的X-ray 結構並不完整,我們將用homology modeling, structural alignment 和 rotamer/fold libraries 來填補這些未解析出來的原子,片斷或整個結構蛋白。新的結構模型經過能量最小化及動力學模擬將使結構在能量上更為合理。我們接著用彈性網路模型和線性響應理論去描述此結構固有的(intrinsic)及擾動後的(perturbed)動力學特徵。此外也將對刺激子和減弱子的結構穩定性及摺疊動力學做模擬。
Abstract: The long-term goal of this interdisciplinary project is to explore the functions and mechanism of ribosomal components during translation, by using advanced single-molecule techniques in combination with biochemical, biophysical and computational methods. In this proposal, we plan to explore the impacts of mRNA structure unfolding and refolding during translation on the ribosomal helicase activity and -1 ribosomal frameshifting regulation to help elucidating the mechanism of ribosomal unwinding activity. Subproject 1 will use biochemical techniques to track the unfolding and refolding processes of ribosome-bound RNA structural motif capable of modulating -1 PRF efficiency. Among them, the Shape analysis will follow the RNA conformations of different ribosome-associated RNA and UV crosslinking will help identifying ribosomal components involved in this process. These information will then be combined with those obtained in the single-molecule approach in subproject 2 and be used in subproject 4 to build a model to describe this novel regulation mechanism of -1 PRF. Subproject 2 will explore the RNA structural stability and dynamics in the presence or absence of ribosome using single-molecule approaches. The stimulator hTPK-DU177 and its mutants will be mechanically unfolded by optical tweezers to determine if the unfolding force is correlated with their capability to stimulate frameshifting. Responses of the RNA stimulators and attenuators under the action of ribosome will be measured on optical tweezers as well as by single-molecule FRET. The data will provide clues on how RNA structural dynamics plays a role in ribosomal frameshifting. Subproject 3 will use NMR to determine the structures and dynamics of a number of related RNA pseudoknots revealed in subproject 1. Particularly, structural dynamic information regarding with the variants of hTPK-ΔU177 will be studied by NMR hydrogen-deuterium exchange (HDX) analysis and spin relaxation dynamics and relaxation dispersion approaches. In addition, allostery and folding cooperativity in these structures will also be explored。Subproject 4 will use computer simulations and proper physical models to analyze mechanical properties of ribosome and unravel possible mechanisms of allosteric regulations promoting/deteriorating -1 programmed ribosomal frameshifting (-1 PRF). The X-ray-solved existing ribosome structures contain unresolved missing segments/subunits. We take the most complete structure to date from T thermophilus as template and mend the structure by homology modeling, structural alignment and de novo modeling using rotamer/fold libraries. We then use elastic network model and linear response theory to analyze the intrinsic and perturbed dynamics of this energetically relaxed new structure. We will also perform unfolding and folding studies on stimulators and attenuators, respectively, using MD simulations. Finally, the theoretical results derived from steered-MD-based mechanical unfolding and thermal unfolding simulations will be compared with the optical tweezer characterized unfolding forces and -1 PRF efficiency. Folding studies of attenuator will also benefit from the NMR H/D exchange data, order parameter data and chemical reactivity of 2’ hydroxyl-group of nucleotides to predict its most dominant conformation as well as dynamics. The predictions will also provide a guidance to further experimental designs in the pursue of -1 PRF mechanism.
Keyword(s)
-1 轉譯框架移轉
核醣體解旋功能
單分子光鉗
核磁共振光譜
計算生物學
-1 ribosomal frameshifting
ribosomal helicase
single-molecule
NMR
computational biology