研究生: |
李承芳 Cheng-Fang Li |
---|---|
論文名稱: |
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. |
指導教授: |
黎耀基
Lai, Yiu-Kay 杜鎮 Tu, Jenn |
口試委員: |
黎耀基
Lai, Yiu-Kay 譚婉玉 Tarn, Woan-Yuh 吳惠南 Wu, Huey-Nan 杜鎮 Tu, Jenn |
學位類別: |
博士 Doctor |
系所名稱: |
生命科學暨醫學院 - 生物科技研究所 Biotechnology |
論文出版年: | 2011 |
畢業學年度: | 99 |
語文別: | 英文 |
論文頁數: | 249 |
中文關鍵詞: | 第二型內含子 、核酸脢結構 、RNA的結構轉換 、DVI 結合位置 |
外文關鍵詞: | group II intron, ribozyme structure, conformational rearrangements, docking site of DVI, allosteric ribozyme |
相關次數: | 點閱:3 下載:0 |
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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。
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.
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.
Boulanger, S. C., P. H. Faix, et al. (1996). "Length changes in the joining segment between domains 5 and 6 of a group II intron inhibit self-splicing and alter 3' splice site selection." Mol Cell Biol 16(10): 5896-904.
Brion P, Westhof E (1997). "Hierarchy and dynamics of RNA folding". Annu Rev Biophys Biomol Struct 26: 113–37.
Cech TR.(2000). ”Structural biology. The ribosome is a ribozyme”. Scienc 289(5481):878-9.
Chanfreau G, Jacquier A (1994) Catalytic site components common to both splicing steps of a group II intron. Science. 266(5189):1383-7.
Chanfreau G, Jacquier A (1996) An RNA conformational change between the two chemical steps of group II self-splicing. EMBO J. 15(13):3466-76.
Chin K, Pyle AM ( 1995) Branch-point attack in group II introns is a highly reversible transesterification, providing a potential proofreading mechanism for 5'-splice site selection. RNA. 1(4):391-406.
Chu, V. T., Q. Liu, et al. (1998). "More than one way to splice an RNA: branching without a bulge and splicing without branching in group II introns." RNA 4(10): 1186- 202.
Chu, V. T., C. Adamidi, et al. (2001). "Control of branch-site choice by a group II intron." EMBO J 20(23): 6866-76.
Copertino DW, Hallick RB (1993) Group II and group III introns of twintrons: potential relationships with nuclear pre-mRNA introns. Trends Biochem Sci. 18(12):467-71.
Costa, M. and F. Michel (1995). "Frequent use of the same tertiary motif by self- folding RNAs." EMBO J 14(6): 1276-85.
Costa M, Deme E, Jacquier A, Michel F (1997) Multiple tertiary interactions involving domain II of group II self-splicing introns. J Mol Biol. 267(3):520-36.
Costa M, Fontaine JM, Loiseaux-de Goer S, Michel F (1997) A group II self-splicing intron from the brown alga Pylaiella littoralis is active at unusually low magnesium concentrations and forms populations of molecules with a uniform conformation. J Mol Biol. 274(3):353-64.
Costa M, Michel F, Westhof E (2000) A three-dimensional perspective on exon binding by a group II self-splicing intron. EMBO J. 19(18):5007-18.
Costa M, Michel F, Toro N (2006) Potential for alternative intron-exon pairings in group II intron RmInt1 from Sinorhizobium meliloti and its relatives. RNA. 12(3):338-41.
Cousineau, B., D. Smith, et al. (1998). "Retrohoming of a bacterial group II intron: mobility via complete reverse splicing, independent of homologous DNA recombination." Cell 94(4): 451-62.
Dai, L., D. Chai, et al. (2008). "A three-dimensional model of a group II intron RNA and its interaction with the intron-encoded reverse transcriptase." Mol Cell 30(4): 472- 85.
Daniels DL, Michels WJ Jr, Pyle AM (1996) Two competing pathways for self- splicing by group II introns: a quantitative analysis of in vitro reaction rates and products. J Mol Biol. 256(1):31-49.
De la Pena, M. and I. Garcia-Robles (2010) "Ubiquitous presence of the hammerhead ribozyme motif along the tree of life." RNA 16(10): 1943-50.
Edgell, D. R., M. Belfort, et al. (2000). "Barriers to intron promiscuity in bacteria." J Bacteriol 182(19): 5281-9.
Fedorova, O., T. Mitros, et al. (2003). "Domains 2 and 3 interact to form critical elements of the group II intron active site." J Mol Biol 330(2): 197-209.
Fedorova, O. and A. M. Pyle (2005). "Linking the group II intron catalytic domains: tertiary contacts and structural features of domain 3." EMBO J 24(22): 3906-16.
Fedorova O, Zingler N. (2007) Group II introns: structure, folding and splicing mechanism. Biol Chem. 388(7):665-78.
Gordon PM, Piccirilli JA (2001) Metal ion coordination by the AGC triad in domain 5 contributes to group II intron catalysis. Nat Struct Biol. 8(10):893-8.
Granlund, M., F. Michel, et al. (2001). "Mutually exclusive distribution of IS1548 and GBSi1, an active group II intron identified in human isolates of group B streptococci." J Bacteriol 183(8): 2560-9.
Hamill S, Pyle AM (2006) The Receptor for Branch-Site Docking within a Group II Intron Active Site. Mol Cell. 23(6):831-40.
Jacquier A, Jacquesson-Breuleux N (1991) Splice site selection and role of the lariat in a group II intron. J Mol Biol. 219(3):415-28.
Jacquier A, Michel F (1987) Multiple exon-binding sites in class II self-splicing introns. Cell. 50(1):17-29.
Jacquier A, Michel F (1990) Base-pairing interactions involving the 5' and 3'-terminal nucleotides of group II self-splicing introns. J Mol Biol. 213(3):437-47.
Jacquier A (1996) Group II introns: elaborate ribozymes. Biochimie. 78(6):474-87.
Jarrell KA, Peebles CL, Dietrich RC, Romiti SL, Perlman PS. (1988)Group II intron self-splicing. Alternative reaction conditions yield novel products.
Karberg M, Guo H, Zhong J, Coon R, Perutka J, Lambowitz AM (2001) Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nat Biotechnol. 19(12):1162-7.
Koonin EV (2006) The origin of introns and their role in eukaryogenesis: a compromise solution to the introns-early versus introns-late debate? Biol Direct. 1:22.
Koch JL, Boulanger SC, Dib-Hajj SD, Hebbar SK, Perlman PS (1992) Group II introns deleted for multiple substructures retain self-splicing activity. Mol Cell Biol. 12(5):1950-8.
Lambowitz AM, Zimmerly S. (2004) Mobile group II introns. Annu Rev Genet. 38:1- 35.
Lehmann K, Schmidt U (2003) Group II introns: structure and catalytic versatility of large natural ribozymes. Crit Rev Biochem Mol Biol. 38(3):249-303.
Michel F, Umesono K, Ozeki H (1989). Comparative and functional anatomy of group II catalytic introns--a review. Gene. 82(1):5-30.
Michel F, Ferat JL (1995) Structure and activities of group II introns. Annu Rev Biochem. 64:435-61.
Michel, F., M. Costa, et al. (2009). "The ribozyme core of group II introns: a structure in want of partners." Trends Biochem Sci 34(4): 189-99.
Mohr, G., D. Smith, et al. (2000). "Rules for DNA target-site recognition by a lactococcal group II intron enable retargeting of the intron to specific DNA sequences." Genes Dev 14(5): 559-73.
Mullineux, S. T., M. Costa, et al. "A group II intron encodes a functional LAGLIDADG homing endonuclease and self-splices under moderate temperature and ionic conditions." RNA 16(9): 1818-31.
Nissen, P., Hansen, J., Ban, N., Moore, P.B., Steitz, T.A. (2000). “The structural basis of ribosome activity in peptide bond synthesis.” Science 289(5481):920-30.
Pley, H. W., D. S. Lindes, et al. (1994). "Crystals of a hammerhead ribozyme." J Biol Chem 269(6): 4692.
Podar M, Perlman PS, Padgett RA. (1998) The two steps of group II intron self- splicing are mechanistically distinguishable. RNA. Aug;4(8):890-900.
Podar, M., J. Zhuo, et al. (1998). "Domain 5 binds near a highly conserved dinucleotide in the joiner linking domains 2 and 3 of a group II intron." RNA 4(2): 151-66.
Pyle, A. M., O. Fedorova, et al. (2007). "Folding of group II introns: a model system for large, multidomain RNAs?" Trends Biochem Sci 32(3): 138-45.
Pyle, A. M. "The tertiary structure of group II introns: implications for biological function and evolution." Crit Rev Biochem Mol Biol 45(3): 215-32.
Qin, P. Z. and A. M. Pyle (1998). "The architectural organization and mechanistic function of group II intron structural elements." Curr Opin Struct Biol 8(3): 301-8.
Reiter NJ, Osterman A, Torres-Larios A, Swinger KK, Pan T, Mondragón A. (2010). The structure of the entire RNAseP holoenzyme -tRNA complex has been solved: Nature. 9;468(7325):784-9.
Rest JS, Mindell DP (2003) Retroids in archaea: phylogeny and lateral origins. Mol Biol Evol. 20(7):1134-42.
Roitzsch M, Pyle AM. (2009) The linear form of a group II intron catalyzes efficient autocatalytic reverse splicing, establishing a potential for mobility. RNA. 15(3):473- 82. Epub 2009 Jan 23.
San Filippo J, Lambowitz AM (2002) Characterization of the C-terminal DNA- binding/DNA endonuclease region of a group II intron-encoded protein. J Mol Biol. 324(5):933-51.
Schmidt, U., M. Podar, et al. (1996). "Mutations of the two-nucleotide bulge of D5 of a group II intron block splicing in vitro and in vivo: phenotypes and suppressor mutations." RNA 2(11): 1161-72.
Sigel, R. K., A. Vaidya, et al. (2000). "Metal ion binding sites in a group II intron core." Nat Struct Biol 7(12): 1111-6.
Sigel RK, Sashital DG, Abramovitz DL, Palmer AG, Butcher SE, Pyle AM (2004) Solution structure of domain 5 of a group II intron ribozyme reveals a new RNA motif. Nat Struct Mol Biol. 11(2):187-92.
Stoddard BL (2005) Homing endonuclease structure and function. Q Rev Biophys. 38(1):49-95.
Su, L. J., C. Waldsich, et al. (2005). "An obligate intermediate along the slow folding pathway of a group II intron ribozyme." Nucleic Acids Res 33(21): 6674-87.
Suchy M, Schmelzer C (1991) Restoration of the self-splicing activity of a defective group II intron by a small trans-acting RNA. J Mol Biol. 222(2):179-87.
Toor, N., G. Hausner, et al. (2001). "Coevolution of group II intron RNA structures with their intron-encoded reverse transcriptases." RNA 7(8): 1142-52.
Toor N, Zimmerly S (2002) Identification of a family of group II introns encoding LAGLIDADG ORFs typical of group I introns. RNA. 8(11):1373-7.
Toor N, Robart AR, Christianson J, Zimmerly S (2006) Self-splicing of a group IIC intron: 5' exon recognition and alternative 5' splicing events implicate the stem-loop motif of a transcriptional terminator. Nucleic Acids Res. 34(22):6461-71.
Toor, N., K. S. Keating, et al. (2008). "Crystal structure of a self-spliced group II intron." Science 320(5872): 77-82.
Toor, N., K. S. Keating, et al. (2010). "Tertiary architecture of the Oceanobacillus iheyensis group II intron." RNA 16(1): 57-69.
Vogel J, Borner T (2002) Lariat formation and a hydrolytic pathway in plant chloroplast group II intron splicing. EMBO J. 21(14):3794-3803.
Watanabe, K. and A. M. Lambowitz (2004). "High-affinity binding site for a group II intron-encoded reverse transcriptase/maturase within a stem-loop structure in the intron RNA." RNA 10(9): 1433-43.
Wheelan, S. J., Y. Aizawa, et al. (2005). "Gene-breaking: a new paradigm for human retrotransposon-mediated gene evolution." Genome Res 15(8): 1073-8.
Wicker, T., Sabot, F., Hua-Van, A., Bennetzen, J.L., Capy, P., Chalhoub, B., Flavell, A., Leroy, P., Morgante, M., Panaud, O., et al. (2007). "A unified classification system for eukaryotic transposable elements". Nat Rev Genet 8: 973-982.
Winkler WC, Nahvi A, Roth A, Collins JA, Breaker RR. 2004. Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428: 281–286.).
Xiang, Q., P. Z. Qin, et al. (1998). "Sequence specificity of a group II intron ribozyme: multiple mechanisms for promoting unusually high discrimination against mismatched targets." Biochemistry 37(11): 3839-49.
Yang, J., Zimmerly, S., Perlman, P.S., Lambowitz, A.M. (1996). "Efficient integration of an intron RNA into double-stranded DNA by reverse splicing". Nature 381(6580):332-5.
Zimmerly, S., Guo, H., Eskes, R., Yang, J., Perlman, P.S., Lambowitz, A.M. (1995). "A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility". Cell 83(4):529-38
Zimmerly S, Moran JV, Perlman PS, Lambowitz AM (1999) Group II intron reverse transcriptase in yeast mitochondria. Stabilization and regulation of reverse transcriptase activity by the intron RNA. J Mol Biol. 289(3):473-90.