Members of the ubiquitous GTPase superfamily regulate numerous cellular functions. A core group of eight GTPases are present in all domains of life: initiation factor 2, elongation factors Tu and G, protein secretion factors Ffh and FtsY, and the poorly characterized factors YihA, YchF, and HflX. While the first five members have well defined roles in the essential cellular process of protein synthesis, a role for YihA, YchF and HflX in this process has only recently been suggested. Here, a detailed kinetic analysis examining the interaction between HflX and its cellular partners is described. 50S and 70S ribosomal particles function as GTPase activating factors for HflX by stabilizing the nucleotide binding pocket of HflX, inducing a “GTPase activated” state. These data indicates a novel mode of GTPase activation, and suggests a role for HflX in regulating translation. / xii, 185 leaves : ill. (some col.) ; 28 cm

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

LepA is a highly conserved GTP-binding protein of unknown function. Its amino acid sequence reveals that it is a GTPase with homology to elongation factor G (EF-G). Previous data led to the hypothesis that LepA negatively regulates a posttranslational process such as protein folding. To examine this possibility, two sets of strains carrying mutated alleles encoding molecular chaperones in E. coli were transformed with a lepA expression vector. LepA had a dominant negative effect specifically in a dnaK25 strain whose product exhibits a 20-fold lower ATPase activity compared to wild-type DnaK. The expression of DnaK and other heat-shock proteins is repressed following temperature downshift. Aptly, it was found that temperature shift from 37 degrees Celcius to 15 degrees Celcius in cells harboring a lepA expression vector led to the induction of lepA and downstream lepB. Furthermore, like cold-shock genes, lepA and lepB are induced by sublethal doses of chloramphenicol, although it appears that lep operon induction is related to the antibiotic's action on the 50S ribosome. Due to LepA's insolubility, it could not be confirmed whether it interacts with DnaK, DnaJ or which other proteins it interacts with. Two-dimensional gel electrophoretic analysis revealed the absence of an isoform of OmpA in two lepA deletion strains. It is possible that LepA is involved in a folding pathway that is responsible for the conformation of this isoform. Phylogenetic analysis showed that while LepA is extremely well conserved and has been identified in all completed Bacterial and Eukaryal genomes, it is not present in the completed genomes of any Archaea. Sequence analysis revealed the existence of N-terminus mitochondrial import sequences in Eukaryal LepA orthologues. Additionally, A. thaliana contains a second LepA orthologue that clusters phylogenetically with Synechocystis LepA and has a chloroplastic import sequence. This indicates that plastidal LepA was acquired in A. thaliana (and probably in all plants) through endosymbiosis of an ancestral cyanobacterium. In constrast, mitochondrial LepA are not closely related to those of a- proteobacteria, believed to be the precursors of mitochondria. These findings imply that in sharp contrast to mitochondrial LepA, chloroplastic LepA is under strong evolutionary pressure to remain conserved.

The objective of this thesis was to synthesize N2-modified guanosines (N2G) in order to introduce fluorescent and chelating ligands, such as diphenylamino, 2,2’-dipridylamino, 2-(2’-pyridyl)benzimidazolyl and p-pyrenylphenyl functionalities. Their photophysical properties were examined in order to gain further knowledge about the effect of guanine modification on its electronic structure. The impact of N2-modification was first studied in terms of self-assembly of the luminescent guanosines in solution and gas phase. Extensive NMR and ESI MS studies provided evidence that these N2-modified guanosines self-assemble exclusively into octamers with high-fidelity, in the presence of Group 1 and Group 2 metal ions. In addition, the first example of “empty” G-octamer (free of metal cations) was identified by ESI MS. Experimental results suggested that N2-substituents provide additional electronic and steric effects which drive the diastereoselectivity of self-assembly and provide additional stability. Hydrogen bonding of N2Gs with cytidine was monitored using fluorescence and NMR. In addition to GC base pair formation, the G-quartet-to-GC base pair structural transformation was studied using CD, fluorescence, and NMR spectroscopy. Due to the luminescent and chelating nature of some of the N2G derivatives, their interactions with a number of metal ions, such as Zn2+, Cd2+, Hg2+, La3+, Tb3+ and Eu3+ ions, were probed by using various spectroscopic methods. The overall optical response in the presence of metal ions was highly dependent on the nature of N2-substituent, and it varied from “turn-on” to “turn-off” response, clearly indicating that the modification at the N2-site of guanosine can be used to finely tune the optical response of these nucleosides. Finally, synthesis of a phosphorescent N2-arylguanosine containing the Ru2+ metal center was achieved and its photophysical and electrochemical properties were examined. / Thesis (Ph.D, Chemistry) -- Queen's University, 2009-09-22 16:02:20.934

Thesis (Ph. D.)--University of New South Wales, 2001. / Also available online.

Thesis (M.S.)--Montana State University--Bozeman, 2005. / Typescript. Chairperson, Graduate Committee: Algirdas J. Jesaitis. Includes bibliographical references (leaves 75-80).

This thesis is divided into three main sections. The first chapter contains a brief review of nitrogen heterocyclic chemistry. The second chapter reports the results and their discussion of new heterocyclic chemistry, and the experimental details are provided in the fourth chapter.

Several short oligoribonucleotide sequences containing modified purine residues of biological significance were synthesized using the phosphotriester method developed in Neilson's laboratory. Variable temperature proton nuclear magnetic resonance (NMR) spectroscopy was used to examine the solution conformations of these oligomers. Studies of the effect of position, type and extent of alkyl modifications on helix structure and stability were undertaken. The triribonucleotide GpCpA was the first trimer shown to form a stable RNA duplex (Tm 33°C) (Alkema, et al, 1981(a)). This duplex contained two G:C base pairs and two 3'-dangling adenosine residues, and had a stability equal to that of the tetramer duplex UpGpCpA, having four Watson Crick base pairs. A series of GpCpN trimers was prepared (N= m⁶A, m⁶₂A, m¹G, m⁶G, e⁶G, m²m⁶G), using GpCpA as a reference, to determine how N-or O-alkylation of the dangling residue affected duplex stability. Both the studies of the N-alkylated (N=m⁶A, m⁶₂A, m¹G) (D'Andrea, et al, 1983) and 0-alkylated (N=m⁶G, e⁶G, m²m⁶G) sequences (present work) led to the conclusion that site and degree of modification were important factors for stability. Comparison of N-versus O-alkylated sequences revealed how the hydrophobic regions surrounding the alkylated nitrogen or alkylated oxygen atoms, and the spatial location of these regions, contributed to duplex stability. Examination of the effect of modified guanosine residues within a short squence, was performed through studies on ApGpNpCpU pentamers (N=m⁶G, e⁶G, m²m⁶G), having N in internal non-base-pairing and. in internal base-pairing positions. No duplex formation was seen, in contrast to studies involving reference compounds : ApGpGpCpU (a qualitative reference), ApGpGpCpU : ApGpUpCpU (Tm 31.4 C), and ApGpGpCpU : ApGpCpCpU (Tm 47.0 C). As no melting temperatures could be calculated for the modified strands, and because their NMR analyses were so similar, no comparisions regarding degree of destabilization, could be made amongst the various modified residues. Never the less, it is clear that O-alkylation of the central G residue significantly disrupts duplex formation, through generation of a centre of great instability. This result sharply contrasts that when the same modified residues are located in terminal, non-bonding positions. / Thesis / Master of Science (MSc)

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