第二型內含子核酸脢的立體結構與結構轉換之研究

Structure and conformational rearrangements during splicing of the ribozyme component of group II introns.

李承芳(Cheng-Fang Li)

指導教授 ： 黎耀基 ； 杜鎮

國立清華大學/生命科學院/生物科技研究所/博士(2011年)

https://doi.org/10.6843/NTHU.2011.00263

摘要

第二型內含子(Goup II intron)為一種能在不需要任何蛋白質幫助的情況 下,能自我催化方式進行剪接(self-splicing)的RNA脢(Ribozyme)。在二級結構 上第二型內含子可以區分為六個保守區塊(Domain),分別為Domain I-VI,在 構築三級結構的過程中,每個保守區塊各有其功能性。各保守區塊利用彼此 分子間的交互作用力構築出類似蛋白質的二級與三級結構,並且利用Domain VI上突出的adenine核苷酸能(Bulge A)與5 端的guanine核苷酸進行兩步驟的催化反應。由於第二型內含子在構造上與真核生物的剪接體(spliceosome)有演化的親緣關係,因此第二型內含子三級結構模型的建立對於了解剪接體的 演化與作用機制有莫大的幫助。然而,儘管第二型內含子的研究從發現到現 在已經有十幾年了,許多第二型內含子分子間的三級作用力與X-ray繞射模型 已經陸續的被發表了,不過關於最重要的Domain VI的結合位置(docking site) 與其周邊的構造一直沒有被成功的解讀。

藉由大規模的親緣關係分析與分子模型推算,我們成功的發現了數個位 於Domain I的區塊,有可能作為Domain VI與branch-point adenosine的連接 處,利用分子突變與選殖的技巧,我們成功的創造出數個突變株,配合動力 學的分析方法,我們發現了兩個位於Domain IC1上的特定核苷酸極有可能就 是Domain VI的結合位置。進一步,我們利用額外加入的小分子核酸 (oligonucleotides)作為鎖鏈(Tether)來牽引失去正常功能的Domain VI去接近潛 在的接合位置。藉由此人工的核酸分子,我們成功的引導了缺失Domain VI 去回復正確的接合功能,也證明了我們假設的接合位置的正確性,這也是目 前首次利用額外添加的核酸分子去引導核酸結構的實驗。藉由這個實驗的成 果,我們成功的建立了一個在Domain VI在第一階段轉酯化反應的原子等級 (atomic-resolution)模型,也更進一步的闡述了第二內含子在催化過程中的分子轉換模型。

關鍵字

第二型內含子 ； 核酸脢結構 ； RNA的結構轉換 ； DVI 結合位置

並列摘要

Group II introns are a class of RNAs best known for their ribozyme- catalyzed, self-splicing reaction. Under certain conditions, the introns can excise themselves from precursor mRNAs and ligate together their flanking exons, without the aid of proteins. Group II introns generally excise from pre-mRNA as a lariat, like the one formed by spliceosomal introns, similarities in the splicing mechanism suggest that group II introns and nuclear spliceosomal introns may share a common evolutionary ancestor.
Despite their very diverse primary sequences, group II introns are defined by a highly conserved secondary structure. This generally consists of six domains (Domain I-Domain VI; D1-D6) radiating from a central wheel. Each of the six intronic domains has a specific role in folding, conformational rearrangements or catalysis. The native conformation of a group II intron is sustained by intra- and interdomain long-range tertiary interactions, which are critical either for folding of the intron to the native state or for its catalytic activity. In brief, Domain V interacts with Domain I to form the minimal catalytic core; Domain VI contains a highly conserved bulged adenosine serving as the branch-point nucleotide. DII and Domain III contribute to RNA folding and catalytic efficiency. Domain IV, which encodes the intron ORF, is dispensable for ribozyme activity.
Group II intron splicing proceeds through two-step transesterification reactions which yield ligated exons and an excised intron lariat. It is initiated by the 2’-hydroxyl group of the bulged adenosine within Domain 6, which serves as a branch point and attacks the phosphate at the 5’-end of the intron, thus releasing the 5’-exon while forming a lariat structure in the first step. The released 5’-exon, which is bound to the intron through base pairing interactions, is then positioned correctly to attack the 3’- splice site with its free 3’-OH in the second step of splicing.
It is generally believed that the structure of a group II ribozyme undergoes conformational rearrangements between first step and second step and domain VI must play a central role in the process. However, despite the identification of several interdomain tertiary interactions, neither NMR nor chemical probing studies have been successful in determining the local surroundings of the branch-point adenosine and neighboring domain VI nucleotides in the ribozyme active site.
By using phylogenetic analysis and molecular modelling, we have identified several areas of the molecule which have the potential to constitute the docking site of domain VI. Mutations were introduced in putative binding sites and the resulting, mutant RNAs have been kinetically characterized. This has allowed us to identify a site within the ribozyme that appears to be specifically involved in the branching reaction. In order to further investigate the interaction between that site and domain VI, we set up a system in which the docking of domain VI into its presumed binding site is ensured by the addition of DNA/RNA oligos that position the two RNA elements in an appropriate orientation. By combining the information from such experiments, we have built an atomic-resolution model of the complex formed by domain VI, the branch site and the rest of the intron at the time at which splicing is initiated.

並列關鍵字

group II intron ； ribozyme structure ； conformational rearrangements ； docking site of DVI ； allosteric ribozyme

參考文獻

Bassi, G. S., D. M. de Oliveira, et al. (2002). "Recruitment of intron-encoded and co- opted proteins in splicing of the bI3 group I intron RNA." Proc Natl Acad Sci U S A 99(1): 128-33.

Bassi, G. S. and K. M. Weeks (2003). "Kinetic and thermodynamic framework for assembly of the six-component bI3 group I intron ribonucleoprotein catalyst." Biochemistry 42(33): 9980-8.

Belhocine K, Yam KK, Cousineau B (2005) Conjugative transfer of the Lactococcus lactis chromosomal sex factor promotes dissemination of the Ll.LtrB group II intron. J Bacteriol. 187(3):930-9.

Boudvillain, M. and A. M. Pyle (1998). "Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis." EMBO J 17(23): 7091-104.

Boudvillain M, de Lencastre A, Pyle AM (2000) A tertiary interaction that links active-site domains to the 5' splice site of a group II intron. Nature. 406(6793):315-8.