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研究生:石仲浩
研究生(外文):Zhonghao Shi
論文名稱:多核苷酸磷酸酶在核醣核酸結合與分解之結構與功能研究
論文名稱(外文):Structural and functional studies of polynucleotide phosphorylase in RNA binding and degradation
指導教授:翟建富翟建富引用關係袁小琀
指導教授(外文):Kin-Fu ChakHanna S. Yuan
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:85
中文關鍵詞:多核苷酸磷酸酶蛋白質晶體結構
外文關鍵詞:polynucleotide phosphorylasecrystal structure
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論文摘要
在生物體內,降解核醣核酸(RNA degradation)是一重要的生理活動,調控著許多細胞生長所必須生理機能,如基因的表現、對各種核醣核酸之生合成(biogenesis)進行品質控制(quality control),甚至可幫助細胞抵抗病毒入侵。在細菌體中,多核苷酸磷酸脢(polynucleotide phosphorylase, PNPase)是一參與核醣核酸分解的重要的核醣核酸外切脢(exoribonuclease),可從單股的核醣核酸之3’端往5’端逐一進行分解作用,在分解訊息核醣核酸(mRNA)方面扮演重要角色。在此研究中,我們分析了大腸桿菌(Escherichia coli)和嗜高溫細菌(Geobacillus kaustophilus)之多核苷酸磷酸脢的生化特性和晶體三維結構,以作為了解多核苷酸磷酸脢在分解訊息核醣核酸的基礎。
多核苷酸磷酸脢具有四個主要的功能區(domain),其N端有兩個RNase PH功能區,其C端則有KH/S1功能區。首先為了解KH/S1功能區在多核苷酸磷酸脢中之功能,我們建構並純化出大腸桿菌之全長以及切除C端KH/S1功能區之多核苷酸磷酸脢的蛋白質,並由活性分析以及凝膠電泳遷移率變動分析(EMSA),證明多核苷酸磷酸脢之C端KH/S1功能區會參與核醣核酸受質之結合,其降解核醣核酸之活性也因與核醣核酸結合之親和力(affinity)下降而明顯降低。有趣的是缺少KH/S1功能區的多核苷酸磷酸脢在分解20個核醣核酸長的受質時會產生較長的最終產物(10~12個核糖核酸長),並且無法分解8個核糖核酸長的受質。而野生株蛋白質則可完全地分解受質。由此生化特性的改變,可看出KH/S1功能區並不只參與受質結合,可能還有其他未知的功能。更進一步的以圓二色光譜儀(circular dichroism)及動態光散射粒徑分析儀(dynamic light scattering)進行分析後證實,C端KH/S1功能區被切除之多核苷酸磷酸脢所形成之三聚體比之全長的多核苷酸磷酸脢,於較高溫度更為不穩定且更易於酸性溶液中分離成單體(monomer),可見S1/KH功能區亦與多核苷酸磷酸脢三聚體的形成與穩定有關。
為了觀察C端KH/S1功能區被切除後結構是否改變而導致出現不同的酵素特性,我們更進一步的解析出了大腸桿菌之全長以及切除C端KH/S1功能區之多核苷酸磷酸脢的蛋白質晶體結構,其解析度各為2.6 Å 和 2.8 Å。由全長多核苷酸磷酸脢之結構可見多核苷酸磷酸脢之三聚體組成一圓球形構造,並且於中央形成一通道,此通道為核醣核酸受質進入多核苷酸磷酸脢活性區之路徑。而C端KH/S1功能區被切除之多核苷酸磷酸脢的蛋白質晶體結構與全長結構比對後,發現其結構較為疏鬆擴張,其中心通道明顯比較寬大,尤其是靠近活性區的通道。由生化分析與結構測定的實驗結果可證明多核苷酸磷酸脢之C端KH/S1功能區不僅與核醣核酸的結合有關,並且會幫助多核苷酸磷酸脢形成更緊密結實且穩定的三聚體。
另外因為在大腸桿菌多核苷酸磷酸脢的蛋白質晶體結構中,其C端KH/S1功能區因結構不固定而無法觀測。因此我們解析了另一種嗜高溫細菌(Geobacillus kaustophilus)之全長多核苷酸磷酸脢晶體結構,雖然解析度只有4 Å,無法提供更細微的結構資料,但其整體結構仍清楚的顯示出與大腸桿菌多核苷酸磷酸脢結構相似。而且在大腸桿菌多核苷酸磷酸脢結構中,無法觀察到其C端之KH/S1功能區,在嗜高溫細菌之全長多核苷酸磷酸脢結構中可看出其C端KH/S1功能區之相對位置。經由同源性模擬法(Homology modeling)分析,推測三個S1功能區之間會互相接觸作用,使得多核苷酸磷酸脢形成穩定的三聚體。
為了進一步了解中央通道是否擔任RNA結合之功能,我們發現大腸桿菌多核苷酸磷酸脢中央通道中有兩處特別狹窄,並且皆包含有3個鹼性正電氨基酸-精氨酸(arginine),其中Arg102、Arg103位於上方並靠近洞口,Arg106位於下方並靠近活性區。利用定位突變法將此二處之精氨酸改變為丙氨酸(alanine)後,再由活性測試以及凝膠電泳遷移率變動分析(EMSA)等實驗證實此二種突變蛋白質,一者其分解核醣核酸之活性及與核醣核酸之親合力皆變差;一者則導致多核苷酸磷酸脢在分解核醣核酸受質的能力發生缺陷,其反應完之最終產物比野生蛋白質反應完之產物長,更無法分解8個核醣核酸長之受質,其性質與KH/S1功能區切除之蛋白質一樣,無法完全分解受質。由實驗結果推論出,多核苷酸磷酸脢之中央通道兩處狹窄地區之鹼性正電氨基酸,的確擔任與核醣核酸受質結合的功能,可幫住多核苷酸磷酸脢抓住核醣核酸受質並推往活性區進行分解。
本篇論文經由生化特性與晶體結構等實驗,證實了多核苷酸磷酸脢之C端KH/S1功能區,不僅參與核醣核酸受質的結合,也與其穩定的三聚體之形成有關,並調控著多核苷酸磷酸脢之中央通道的大小而影響了多核苷酸磷酸脢之活性。此外也利用點突變的技術來探討中央通道對於酵素活性的影響,發現位於中央通道狹窄處之精氨酸102、103及106等胺基酸的確有與核醣核酸受質結合的功能,並且進一步的將受質推往活性區進行分解。由此可見多核苷酸磷酸脢之所以能逐一的由核醣核酸之3’端分解受質,中央通道中的胺基酸功不可沒。
Abstract
RNA degradation plays important roles in cells, regulating several biological events, including gene expression, quality control of RNA biogenesis and viral defense. Bacterial polynucleotide phosphorylase (PNPase) plays a major role in messenger RNA turnover by the degradation of mRNA from the 3’-to-5’ ends. Here we analyze the biochemical properties and crystal structures of PNPase from Escherichia coli and Geobacillus kaustophilus to provide a molecular basis for PNPase in RNA degradation.
Bacterial PNPase all have two RNase PH domains followed by a C-terminal KH and S1 domain. To elucidate the function of the KH/S1 domain in PNPase, E. coli full-length and C-terminal KH/S1 domain truncated mutant (ΔKH/S1) were constructed and purified. We found that ΔKH/S1 bound and cleaved RNA less efficiently with an eight-fold reduced binding affinity. Interesting, ΔKH/S1 not only had lower RNase activities, it also generated longer final products of ~10-12-mer RNAs. The thermal melting and acid-induced trimer dissociation studies, analyzed by circular dichroism and dynamic light scattering, further showed that ΔKH/S1 formed a less thermal-stable trimer than the full-length PNPase. These results suggest that the KH/S1 domain is not only involved in RNA binding and it is also involved in the stabilization of PNPase trimer.
To find out why ΔKH/S1 mutant had different biochemical properties, we determined the crystal structures of the E. coli full-length PNPase and ΔKH/S1 at resolutions of 2.6 Å and 2.8 Å, respectively. The six RNase PH domains of the trimeric PNPase assemble into a ring-like structure containing a central channel. The structural comparison between full-length PNPase and ΔKH/S1 shows that the structure of ΔKH/S1 is more expanded, containing a slightly wider central channel than that of the wild-type PNPase. This result suggests that the KH/S1 domain helps PNPase to assemble into a more compact trimer, and therefore it regulates the channel size allosterically. The crystal structure of the full-length G. kaustophilus PNPase was determined at 4.0 Å resolution. It revealed the structure of the KH/S1 domain which was disorder in the structure of E. coli PNPase. In combined with homology modeling studies, it suggested that S1 domain interacts with each other to form a stable trimer in PNPase.
Moreover, the crystal structure of the E. coli PNPase showed that the central channel contains two necks with three arginine residues located in the neck regions: Arg102 and Arg103 in the upper neck closer to the channel entrance, and A106 in the lower neck closer to the active site. Single and double mutants were constructed to replace these arginine residues with alanine. The mutated PNPase bound and cleaved RNA less efficiently or generated longer cleaved oligonucleotide products, confirming that these arginines were involved in RNA binding and processive degradation. Taking these results together, we conclude that the constricted central channel and the basic-charged residues in the channel necks of PNPase play crucial roles in trapping RNA for processive exonucleolytic degradation.
Contents
論文摘要……………………………………………………………………………....1
Abstract………………..……………………………………….…………………......3
1. Introduction………………………………………………...………..….….….....5
1.1. PNPase in bacteria and plant chloroplasts is involved in mRNA degradation……………..……………………..………………………….…...6
1.2. Biological functions of mammalian PNPase remain unclear…………….…....8
1.3. Structure of Bacterial PNPase……………………………………..……….….9
1.4. Archaea and eukaryotic exosomes share similar functions to bacterial PNPase…..……………………………………………………...……………10
1.5. Structure of exosomes is similar to PNPase…………………….…………….13
1.6. Specific aims………………………………………………………………….14
2. Materials and Methods…...………………………………………………….…..16
2.1. Cloning, protein expression and purification…………………………………16
2.2. RNA-binding assays…………………………………………………………..18
2.3. RNase activity assays…………………………………………………………18
2.4. Circular dichroism…………………………………………………………….19
2.5. Dynamics light scattering (DLS)……………………………………………...20
2.6. Crystallization………………………………………………………………...20
2.7. Data collection, structure determination and refinement………………...…...21
3. Results……………………………………………………………………...……..23
3.1. Overexpression, purification and crystallization of E. coli PNPase……………………………….………………………………………23
3.2. PNPase is a metal ion dependent enzyme…………………………………….23
3.3. The S1/KH-truncated PNPase binds RNA less efficiently……………………24
3.4. The KH/S1-truncated PNPase cleaves RNA less efficiently………………….25
3.5. KH/S1 domains in PNPase are involved in trimer formation……….………..26
3.6. Crystal structures of E. coli full-length and �寐H/S1 PNPase…………….....27
3.7. PNPase trimer is more loosely packed without KH/S1 domain.......................28
3.8. Arginine residues in the channel neck regions are involved in RNA binding and processing……………….……………………………………………………29
3.9. The Asp509 at RNase PH2 domain is an active-site residue….……………...30
3.10. Crystallization and structural analysis of G. kaustophilus
PNPase…………………………………………………………..……….…..31
3.11. RhlB may bind to the C-terminal KH/S1 domain of PNPase ….……….......32
4. Discussion………………………………………………………..….………….…34
4.1. The KH/S1 domain helps PNPase to form a more stable compact trimer………………..………………………………………………………..34
4.2. A constricted central channel plays a crucial role in RNA binding and processing………………….……………..……………………………….….36
5. Conclusion……………………………………..……………..…………...………38
List of Tables………………………………………..………..…………………...…39
Tabl 1. Exosome core components and cofactor…………………………………..39
Tabl 2. X-ray data collection and refinement statistics for the full-length E. coli PNPase and the KH/S1 domain-truncated PNPase……………………………………………………….……….......…40
Tabl 3. X-ray data collection statistics for the G. K. PNPas…………………….…41
List of Figures…………………………………………………………………….....42
Figure 1-1 PNPase functions both as a RNA polymerase and a 3’-to-5’ exoribonuclease.………….………………….…………………….……….…42
Figure 1-2. Messanger RNA turnover in E. coli.…….….....................................…43
Figure 1-3. Domain structures of bacterial and eukaryotic PNPases……………...44
Figure 1-4. Crystal structure of S. antibioticus PNPase..………………………….45
Figure 1-5. Functions of eukaryotic exosomes.………………………….......……46
Figure 1-6. Structure of human and archaeal exosome.…………………………...47
Figure 1-7. Crystal structure of archaeal exosome in complex with RNA…………………………………...………………………...…………..48
Figure 3-1. SDS-PAGE analysis for all the purified proteins..................................49
Figure 3-2. Crystals of E. coli PNPase and G. K. PNPase…….……………......…50
Figure 3-3. PNPase is a metal ion-dependent enzyme.……………………..….….51
Figure 3-4. RNA-binding assays for E. coli full-length and ΔKH/S1 PNPase………………...……………….……………….………………...…52
Figure 3-5. RNA digestion activities of the E. coli full-length PNPase and KH/S1-domain truncated mutant.………….……….…………………..…...53
Figure 3-6. Circular dichroism analysis of the E. coli full-length PNPase and KH/S1-domain truncated mutant…………………………………...…….....54
Figure 3-7. Dynamic light scattering (DLS) analysis of the E. coli full-length PNPase and KH/S1-domain truncated mutant................................................55
Figure 3-8. Crystal structure of E. coli. PNPase.…………………...………..……56
Figure 3-9. Structure superposition of E. coli full-length PNPase, ΔKH/S1 PNPase and S. antibioticus PNPase monomer..........................................................57
Figure 3-10. Three arginine residues are located in the neck regions in the central channel of E. coli PNPase..………………………………….……….....…...58
Figure 3-11. Comparison of the crystal structures and buried surfaces between full-length PNPase and truncated mutant ΔKH/S1……………………...…..59
Figure 3-12. Comparison of the central channel between full-length PNPase and ΔKH/S1 mutant…………………….………………………………………..60
Figure 3-13. RNA binding and degradation activities of wild-type and mutated PNPase……………...………………………………………………….……61
Figure 3-14. Structure alignment of E. coli PNPase RNase PH2 domain and archaeal exosome component Rrp41…………...……………..…………….62
Figure 3-15. RNA binding and degradation activities of E. coli PNPase mutant D509A…………………………….……………...................................…….63
Figure 3-16. The G. Kaustophilus PNPase is more stable than E. coli PNPase…………………………………………………...…………...……..64
Figure 3-17. RNA binding and degradation activities of G. Kaustophilus PNPase…………………….……………………………………………..….65
Figure 3-18. Crystal structure of the G. Kaustophilus PNPase at 4.0 Å resolution………………………………………….……………………..….66
Figure 3-19. Gel filtration (superdex 200) analysis of E. coli PNPase and RhlB mixture.Figure 1.…………………………………………….…..………..…67
Figure 4-1. The shape of the central channel of two archaeal exosomes………………………………………………………………….…68
Figure 4-2. A model for trimeric Rrp4 cap on the molecular surface of trimeric PNPase…………………….……………………….………………………..69
Figure 4-3. Comparison of the RNA-binding channel in the E. coli PNPase, Streptomyces antibioticus PNPase, archaeal exosomeand human exosome………………………………….…….……………………...…….70
References…………………………………………………………...………………71
Appendix……………………………………………………………………………80
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