以單分子技術分析人類端粒酶RNA偽結結構之摺疊機制

Single-Molecule Analysis on the Folding Mechanism of Human Telomerase RNA Pseudoknot Structure

陳郁庭(Yu-Ting Chen)

指導教授 ： 温進德

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.10775

摘要

人類端粒酶是由451個鹼基的核醣核酸、一種特定的反轉錄酶(human telomerase reverse transcriptase)以及其他相關的蛋白質所組成。在人類端粒酶核醣核酸的5’ 包含了與端粒形成有關的核醣核酸模板(RNA template)，以及和端粒酶活性有關的高度保存(highly conserved)偽結結構(pseudoknot)。
此高度保存的偽結結構包括了2個類髮夾(hairpin)的結構，2個類髮夾互相作用在結構中形成了大凹槽(major-groove)與小凹槽(minor-groove)，在2個凹槽中有5對三重鹼基對(base-triple)，過去利用偽結突變構造 (hTR DU177)的研究指出，偽結結構之穩定度會受到內部三重鹼基對的影響，當三重鹼基對形成，其穩定性相對提高，但至今對於偽結結構是如何折疊而成的仍沒有一定論。
本研究即是藉由單分子技術，以雷射光鉗(optical tweezers)對hTR DU177施加外力，觀察外力與打開長度之間的關係，發現在打開結構的過程中大部分的是先打開其中一個類髮夾結構，只有在少數情況下會打開整個偽結結構，之後再經過更進一步的實驗後，我們推論形成偽結結構的途徑有2種，絕大多數的情形下偽結是以第一類髮夾結構(hairpin 1)存在居多，要形成完整的偽結結構須花較長時間，但是一旦第二類髮夾結構(hairpin 2) 比第一類髮夾結構先形成，則可迅速形成帶有三重鹼基對的偽結結構。

關鍵字

偽結結構 ； 髮夾結構 ； 三重鹼基對 ； 雷射光鉗 ； 單分子

並列摘要

Human telomerase is composed of a 451 nt RNA (hTR) and several proteins, including a specialized reverse transcriptase (hTERT). The 5’ domain of hTR contains a RNA template for telomere synthesis and a highly conserved pseudoknot structure crucial for telomerase activity.
The highly conserved pseudoknot structure has two hairpin (stem-loop) structures. The interaction of two hairpin structures formed a major-groove and a minor-groove, as well as five triple-base pairs between them. Previous experiments have shown that the stability of the pseudokont derived from hTR (hTR DU177) was related to the base triples. However, the mechanism of pseudoknot folding is not well understood.
In this study, we used optical tweezers to measure the unfolding force of a series of hTR DU177-related structures and analyzed the relationship between the force and the extension. Instead of being completely unfolded in one step, we found that only one of the two hairpins within the pseudoknot was formed and unfolded during most of the recordings. Based on further experiments, we propose that the pseudoknot is formed by two alternative pathways: Formation of the two hairpin structures kinetically competes to each other. DU177 often tends to form the stable hairpin 1 rapidly, but proceeds to fold into the complete pseudoknot in a much slower fashion. On the other hand, the RNA occasionally folds into an intact pseudoknot structure quickly via the less preferred hairpin 2 pathway.

並列關鍵字

pseudoknot ； hairpin ； triple-base pair ； optical tweezers ； single-molecule

參考文獻

Abbondanzieri, E. A., W. J. Greenleaf, J. W. Shaevitz, R. Landick and S. M. Block (2005). "Direct observation of base-pair stepping by RNA polymerase." Nature 438(7067): 460-465.

Ashkin, A., J. M. Dziedzic and T. Yamane (1987). "Optical Trapping and Manipulation of Single Cells Using Infrared-Laser Beams." Nature 330(6150): 769-771.

Block, S. M., L. S. Goldstein and B. J. Schnapp (1990). "Bead movement by single kinesin molecules studied with optical tweezers." Nature 348(6299): 348-352.

Bustamante, C. (2008). "In singulo biochemistry: When less is more." Annual Review of Biochemistry 77: 45-50.

Bustamante, C., Y. R. Chemla, N. R. Forde and D. Izhaky (2004). "Mechanical processes in biochemistry." Annu Rev Biochem 73: 705-748.