蛋白質合成的延伸階段由兩個延伸因子推動，而這兩個延伸因子與核糖體的結合點同樣位於核糖體柄的底部。作為GTP酶，這兩個延伸因子本身無活性，需要依賴GTP酶相關中心在適當的時候把他們轉化為活性酶。真核生物的GTP酶相關中心由28S核糖體核糖核酸58個鹼基、P0（P1/P2）₂五聚體蛋白複合體及柄基蛋白eL12組成。由於核糖體柄的動態結構，這個區域在現今的真核生物核糖體結構研究中仍然未能解構，而我們的研究成功判斷出核糖體柄複合結構的特徵。我們確定了穩定P1/P2異源二聚體的相互作用，指出P1/P2異源二聚體比P2同源二聚體擁有較高的構象穩定性。同時我們發現了P1第三螺旋上一個外露的疏水區，對於P1/P2異源二聚體與P0的結合有重要的作用。就此我們決定了P0的兩個脊柱螺旋為P1/P2異源二聚體的結合點。利用同源模擬法及蛋白突變，我們提出了有關核糖體柄結構的新模型。在這個模型中，結合於P0上的兩個異源二聚體以P2/P1：P1/P2序列。我們提出的模型能夠解釋每個P-蛋白對GTP酶活性的不同貢獻，以及P0上兩個P1/P2異源二聚體的功能協同性。這個模型中核糖體柄結構的方向性，最能配合核糖體柄募集延伸因子的功能。基於對核糖體柄的研究，我們進一步研究柄基蛋白eL12並提出初步數據顯示eL12與核糖體柄之間的直接互動。這個研究結果提出，eL12的功能很可能是透過與核糖體柄的直接活動來傳遞結合及激活訊號。我們就GTP酶相關中心的研究補充了對真核生物核糖體結構的研究，加深了對GTP酶相關中心如何推動蛋白質合成的理解。 / The elongation cycle of protein synthesis is driven by two elongation factors that bind to overlapping sites at the base of the ribosomal stalk. Both factors have limited inherent GTPase activity and they rely on the GTPase associated centre to activate GTP hydrolysis at appropriate times during elongation. In eukaryotes, this region consists of a 58-base 28S ribosomal RNA, the P0(P1/P2)₂ pentameric stalk complex and the stalk base protein eL12. Due to the dynamic nature of the ribosomal stalk, this region remains as a missing piece in the high-resolution structural studies of the eukaryotic ribosome. In this work, we have characterized the structural organization of the stalk complex. We have identified the stabilizing interactions within P1/P2 heterodimer and showed that P1/P2 heterodimer is preferred over P2 homodimer due to its higher conformational stability. We have also identified an exposed hydrophobic patch on helix-3 of P1 that is important for anchoring P1/P2 heterodimers to P0 and we havemapped two spine helices on P0 as the binding sites for P1/P2 heteodimer. Based on homology modelling and mutagenesis experiments, we have proposed a new model of the eukaryotic stalk complex where the two heterodimers display a P2/P1:P1/P2 topology on P0. Our model provides an explanation for the difference of GTPase activities contributed by each P-protein and the functional contribution of the hydrophobic loop between the two spine helices of P0. Our model represented the stalk complex in an orientation that is the most effective for recruiting translation factors to their binding sites. As an extension to our studies, we have preliminary data showing direct interaction between eL12 and stalk complex. This is a strong suggestion that eL12 contributes to its functional role by transmitting signal for factor binding and activation through direct interaction with the stalk complex. Our work on the GTPase associated centre has supplemented the structural studies of the eukaryotic ribosome and provided a betterpicture of how the GTPase associated centre contributes to the high efficiency of protein synthesis. / Detailed summary in vernacular field only. / Yu, Wing Heng Conny. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 117-125). / Abstract also in Chinese. / Chapter i. --- Abstract --- p.1 / Chapter ii. --- 摘要 --- p.3 / Chapter iii. --- Acknowledgments --- p.4 / Chapter iv. --- Disclaimer --- p.5 / Chapter v. --- List of figures --- p.6 / Chapter vi. --- Table of Contents --- p.7 / Chapter Chapter 1. --- Project Background and Objectives --- p.10 / Chapter 1.1. --- The ribosome --- p.10 / Chapter 1.1.1. --- Its components: ribosomal RNA and proteins --- p.10 / Chapter 1.1.2. --- Its function: protein translation --- p.12 / Chapter 1.2. --- The GTPase Associated Centre --- p.13 / Chapter 1.2.1. --- P-complex: P0, P1 and P2 --- p.14 / Chapter 1.2.2. --- Stalk base protein: eL12 --- p.16 / Chapter 1.3. --- Project objectives --- p.17 / Chapter 1.3.1. --- Structural organization of the P-complex --- p.18 / Chapter 1.3.2. --- Characterization of the interaction between eL12 and P-complex --- p.19 / Chapter Chapter 2. --- Methods and Materials --- p.20 / Chapter 2.1. --- DNA Techniques --- p.20 / Chapter 2.1.1. --- Agarose gel electrophoresis of DNA --- p.20 / Chapter 2.1.2. --- Sub-cloning --- p.21 / Chapter 2.1.3. --- Site-directed mutagenesis --- p.23 / Chapter 2.2. --- RNA Techniques --- p.24 / Chapter 2.2.1. --- in vitro transcription and purification of RNA --- p.24 / Chapter 2.2.2. --- Agarose gel electrophoresis of RNA --- p.25 / Chapter 2.2.3. --- Electrophoretic mobility shift assay (EMSA) --- p.26 / Chapter 2.3. --- General protein techniques --- p.27 / Chapter 2.3.1. --- Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.27 / Chapter 2.3.2. --- Native PAGE for acidic proteins --- p.28 / Chapter 2.3.3. --- Protein transfer and Western blotting --- p.29 / Chapter 2.4. --- Expression and purification of recombinant proteins --- p.30 / Chapter 2.4.1. --- Preparation of E. coli competent cells --- p.30 / Chapter 2.4.2. --- Transformation and bacterial culture --- p.31 / Chapter 2.4.3. --- Protein extraction by cell lysis. --- p.32 / Chapter 2.4.4. --- Purification of P2 and mutants --- p.33 / Chapter 2.4.5. --- Purification of P1 and mutants --- p.36 / Chapter 2.4.6. --- Purification of His-P0 and mutants --- p.39 / Chapter 2.4.7. --- Purification of P1/P2 heterodimer and mutants --- p.41 / Chapter 2.4.8. --- Reconstitution and purification of P0(P1/P2)₂ complex and mutants --- p.43 / Chapter 2.4.9. --- Purification of eL12 and mutants --- p.44 / Chapter 2.5. --- Preparation of rat Elongation factor 2 (EF-2) --- p.46 / Chapter 2.5.1. --- Preparation of liver lysate --- p.46 / Chapter 2.5.2. --- Purification of rat EF-2 --- p.47 / Chapter 2.6. --- Circular dichroism (CD) spectrometry --- p.49 / Chapter 2.6.1. --- Chemical denaturation --- p.50 / Chapter 2.6.2. --- Thermal denaturation --- p.51 / Chapter 2.7. --- Limited proteolysis --- p.52 / Chapter 2.8. --- Light scattering (LS) experiments --- p.53 / Chapter 2.8.1. --- Size exclusion chromatography coupled with light scattering detection (SEC/LS) --- p.53 / Chapter 2.8.2. --- Dynamic light scattering (DLS) --- p.54 / Chapter 2.9. --- in vitro binding assay using NHS-activated Sepharose --- p.55 / Chapter 2.10. --- Homology modelling --- p.56 / Chapter 2.10.1. --- Sequence alignment --- p.56 / Chapter 2.10.2. --- Modelling using UCSF Chimera built-in Modeller --- p.57 / Chapter 2.10.3. --- Modelling using Modeller scripts --- p.58 / Chapter 2.11. --- Buffers and reagents --- p.60 / Chapter 2.11.1. --- Media for general bacterial culture --- p.60 / Chapter 2.11.2. --- Reagents for DNA and RNA gel electrophoresis --- p.61 / Chapter 2.11.3. --- Reagents for SDS-PAGE and native PAGE --- p.62 / Chapter 2.11.4. --- Reagents for Western blotting --- p.62 / Chapter 2.12. --- Sequences of DNA oligos --- p.64 / Chapter 2.12.1. --- Primers for P1 mutants --- p.64 / Chapter 2.12.2. --- Primers for P0 mutants --- p.65 / Chapter 2.12.3. --- Primers for eL12 and its mutants --- p.66 / Chapter 2.12.4. --- DNA template for in vitro transcription --- p.68 / Chapter Chapter 3. --- Structural Organization of the Eukaryotic Stalk Complex --- p.69 / Chapter 3.1. --- Introduction --- p.69 / Chapter 3.2. --- Results --- p.71 / Chapter 3.2.1. --- Homology modelling of P1/P2 heterodimer --- p.71 / Chapter 3.2.2. --- P1/P2 heterodimer is stabilized by a hydrophobic interface --- p.74 / Chapter 3.2.3. --- Helix-3 of P1 plays a vital role in P-complex formation --- p.78 / Chapter 3.2.4. --- C-terminal tails are not involved in P-complex formation --- p.80 / Chapter 3.2.5. --- Spine helices of P0 are the binding sites for P1/P2 heterodimers --- p.83 / Chapter 3.2.6. --- Homology modelling of the pentameric complex --- p.86 / Chapter 3.3. --- Discussion --- p.89 / Chapter 3.3.1. --- Comparison between homology model and structure of P1/P2 heterodimer --- p.89 / Chapter 3.3.2. --- Biological significance of P2/P1:P1/P2 topology --- p.92 / Chapter 3.4. --- Towards structure determination of P-complex --- p.97 / Chapter Chapter 4. --- Characterization of the interaction between eL12 and P-complex. --- p.99 / Chapter 4.1. --- Introduction --- p.99 / Chapter 4.2. --- Results --- p.100 / Chapter 4.2.1. --- Homology modelling of human eL12 --- p.100 / Chapter 4.2.2. --- Characterization of recombinant eL12 --- p.103 / Chapter 4.2.3. --- eL12 directly interacts with P-complex via its N-terminal residues --- p.106 / Chapter 4.3. --- Discussion --- p.108 / Chapter 4.4. --- Towards structure determination of eL12 --- p.111 / Chapter Chapter 5. --- Conclusion and future work --- p.114 / Chapter 5.1. --- Proposed working mechanism of eukaryotic GTPase Associated Centre --- p.114 / Chapter 5.1.1. --- Anchorage to the ribosome through RNA binding --- p.114 / Chapter 5.1.2. --- P1/P2 heterodimers are bound to P0 in a P2/P1:P1/P2 topology --- p.114 / Chapter 5.1.3. --- eL12 as functional player in the GTPase associated centre. --- p.115 / Chapter 5.2. --- Future work --- p.116 / Chapter vii. --- References --- p.117
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328481 |
| Date | January 2013 |
| Contributors | Yu, Wing Heng Conny., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (125 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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