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"July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 166-177) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Chiu, Yu Hin Teddy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 118-129). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Content --- p.vi / Abbreviations --- p.x / Naming system for mutant proteins --- p.xi / Abbreviation for amino acid --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- What are acidic ribosomal proteins? --- p.1 / Chapter 1.2 --- Why P-proteins are so important? --- p.13 / Chapter 1.3 --- Research objectives --- p.15 / Chapter Chapter 2 --- Materials and Methods --- p.17 / Chapter 2.1 --- List of buffers and media --- p.17 / Chapter 2.1.1 --- Preparation of buffers and media --- p.17 / Chapter 2.1.2 --- Buffers for preparing competent cells --- p.17 / Chapter 2.1.3 --- Media for bacterial culture --- p.17 / Chapter 2.1.4 --- Buffers for nucleic acid electrophoresis --- p.19 / Chapter 2.1.5 --- Buffers for protein electrophoresis --- p.19 / Chapter 2.1.6 --- Buffers for interaction studies using BIAcore 3000 --- p.21 / Chapter 2.2 --- General methods --- p.23 / Chapter 2.2.1 --- Preparation of Escherichia coli (E.coli.) competent cells --- p.23 / Chapter 2.2.2 --- Transformation of Escherichia coli (E.coli.) competent cells --- p.23 / Chapter 2.2.3 --- DNA cloning --- p.24 / Chapter 2.2.3.1 --- DNA cloning by polymerase chain reaction (PCR) --- p.24 / Chapter 2.2.3.2 --- Agarose gel electrophoresis of DNA --- p.25 / Chapter 2.2.3.3 --- Extraction and purification of DNA from agarose gels --- p.25 / Chapter 2.2.3.4 --- Restriction digestion of DNA --- p.25 / Chapter 2.2.3.5 --- Ligation of digested insert and expression vector --- p.27 / Chapter 2.2.3.6 --- Verification of insert by PCR --- p.27 / Chapter 2.2.3.7 --- Mini-preparation of plasmid DNA --- p.28 / Chapter 2.2.4 --- Polyacrylamide gel electrophoresis (PAGE) of protein --- p.29 / Chapter 2.2.4.1 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29 / Chapter 2.2.4.2 --- Tricine SDS-polyacrylamide gel electrophoresis --- p.30 / Chapter 2.2.4.3 --- Native polyacrylamide gel electrophoresis --- p.31 / Chapter 2.2.4.4 --- Commassie brilliant blue staining of proteinin polyacrylamide gel --- p.32 / Chapter 2.2.4.5 --- Zinc Imidazole staining of protein in polyacrylamide gel --- p.33 / Chapter 2.2.5 --- Protein concentration determination --- p.33 / Chapter 2.2.6 --- Expression of recombinant proteins --- p.33 / Chapter 2.2.6.1 --- Expression of recombinant proteins using LB --- p.33 / Chapter 2.2.6.2 --- Expression of recombinant proteins using minimal medium --- p.34 / Chapter 2.2.6.3 --- Harvest and lysis of bacterial cell culture --- p.34 / Chapter 2.3 --- Protein purification --- p.36 / Chapter 2.3.1 --- Purification of ribosomal protein P1 and its deletion mutants --- p.36 / Chapter 2.3.1.1 --- Purification of P1 --- p.36 / Chapter 2.3.1.2 --- Purification of P1ΔC25 --- p.36 / Chapter 2.3.1.3 --- Purification of HisMBP-P1ΔC40 and HisMBP-P1ΔC47 --- p.37 / Chapter 2.3.2 --- Purification of ribosomal protein P2 and its deletion mutants --- p.38 / Chapter 2.3.2.1 --- Purification of P2 --- p.38 / Chapter 2.3.2.2 --- Purification of P2ΔC46 and P2ΔC55 --- p.39 / Chapter 2.4 --- "Preparation and purification of protein complexes formed by P1, P2 and their truncation mutants" --- p.40 / Chapter 2.4.1 --- Preparation of complexes by Co-refolding in urea buffer --- p.40 / Chapter 2.4.1.1 --- Preparation of P1 or P1ΔC25 involved complexes --- p.40 / Chapter 2.4.1.2 --- Preparation of P1ΔC40/ P2ΔC46 and P1ΔC47/ P2ΔC46 --- p.41 / Chapter 2.4.2 --- Preparation of complexes by direct mixing --- p.42 / Chapter 2.5 --- Laser light scattering for the determination of molecular weight of protein and their complexes --- p.43 / Chapter 2.5.1 --- Chromatography mode light scattering experiment (SEC/LS) --- p.43 / Chapter 2.6 --- Interaction study of P1 and P2 using BIAcore 3000 surface plasmon resonance (SPR) biosensor --- p.45 / Chapter 2.6.1 --- Immobilization of P2 onto CM5 sensor chips --- p.45 / Chapter 2.6.2 --- Kinetic measurements of P1 and P2 interaction --- p.46 / Chapter Chapter 3 --- Determination of domain boundaries for dimerization of P1/P2 --- p.46 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Preparation of P1，P2 and their truncation mutants --- p.50 / Chapter 3.2.1 --- Construction of P1 and P2 N-terminal domains (NTDs) --- p.50 / Chapter 3.2.2 --- P1 and its truncation mutants were purified in denaturing condition --- p.53 / Chapter 3.2.3 --- "P2, P2AC46 and P2AC55 were purified" --- p.56 / Chapter 3.3 --- Formation of complexes from P1，P2 and their truncation mutants --- p.59 / Chapter 3.3.1 --- "P1, P2 and their truncation mutants interact to yield protein complexes" --- p.49 / Chapter 3.3.2 --- P1AC47/P2AC46 is the smallest N-terminal domain complex --- p.63 / Chapter 3.4 --- Perturbation of P2 NTD upon binding with P1 --- p.65 / Chapter 3.4.1 --- "1H, 15N 一 HSQC spectrum of P2AC46 changed significantly upon binding with P1" --- p.65 / Chapter 3.4.2 --- P1/P2AC46 prepared by co-refolding and direct mixing give the same HSQC spectra --- p.66 / Chapter 3.5 --- Discussion --- p.69 / Chapter Chapter 4 --- Stochiometry of P1/P2 Complex is revealed by Light scattering --- p.72 / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.2 --- P1 and P2 interact in 1:1 molar ratio --- p.77 / Chapter 4.2.1 --- Purified P2 exists as homo-dimer in solution --- p.77 / Chapter 4.2.2 --- The stochiometry of P1/P2 complex is 1:1 --- p.78 / Chapter 4.3 --- Stochiometries of P1 and P2 truncation mutant complexes varied from the full-length counterparts --- p.81 / Chapter 4.3.1 --- P2AC46 and P2AC55 exist as homo-dimer in solution --- p.81 / Chapter 4.3.2 --- "P1/P2AC46, P1AC25/P2 and P1AC40/P2AC46 retain the hetero-dimeric stochiometry of 1:1" --- p.82 / Chapter 4.3.3 --- P2AC55 involved complexes show a different stochiometry --- p.83 / Chapter 4.4 --- Discussion --- p.87 / Chapter Chapter 5 --- Binding kinetics of P1/P2 complex studied by surface plasmon resonance --- p.92 / Chapter 5.1 --- Introduction --- p.92 / Chapter 5.2 --- Kinetic parameters of P1 and P2 interaction is revealed by surface plasmon resonance --- p.95 / Chapter 5.2.1 --- P2 was coupled to CM5 sensor chip surface for kinetic studies --- p.95 / Chapter 5.2.2 --- Reduction of basal response after the 1st binding of P1 --- p.96 / Chapter 5.2.3 --- P1 induced a great change in response unit than P2 upon binding with immobilized P2 --- p.99 / Chapter 5.2.4 --- Kinetic parameters of P1 and P2 interaction was studied by introducing P1 to the sensor chip surface --- p.101 / Chapter 5.2.5 --- Dissociation constant derived from 1:1 Langmuir binding isotherm --- p.102 / Chapter 5.2.6 --- Dissociation constant derived from responses at equilibrium (Req) --- p.103 / Chapter 5.3 --- Discussion --- p.106 / Chapter Chapter 6 --- Conclusion and discussion of the study --- p.112 / References --- p.118 / Appendix --- p.130

The eubacteria, archaebacteria and eucaryota evolved from a common ancestral state, the progenote, approximately 4,000 million years ago. The archaebacteria flourish in extreme environments, exhibiting unusual macromolecular structures and metabolism of which much has recently been elucidated. Less, however, is known of the genetics of archaebacteria. In order to investigate gene structure, organization, regulation and evolution in the archaebacteria a gene cluster encoding the ribosomal proteins of the GTPase domain was cloned from the extremely halophilic archaebacterium Halobacterium cutirubrum, characterized and compared with the homologous genes and proteins from eubacteria and eucaryota. A clone containing a 5146 basepair insert of genomic Halobacterium cutirubrum NRCC 34001 DNA encoding the GTPase domain ribosomal proteins was characterized and discovered to retain the identical gene order (i.e. L11e, Lie, L10e and L12e) as the homologous Escherichia coli genes and in addition two transcribed upstream open reading frames encoding the potential proteins ORF, of unknown function and NAB, bearing sequence similarity to nucleic acid binding proteins. The predominant transcripts are monocistronic L11e and tricistronic Lie - L10e - L12e transcripts; monocistronic NAB and bicistronic NAB - L11e transcripts are present at reduced levels and the ORF is present as a very rare transcript. Common elements upstream of the transcription initiation sites include the motif TTCGA ... 4-15 bp ... TTAA ... 20-26 bp ... A or G transcription start. The NAB and some of the ORF transcripts are divergently transcribed from a single TTAA promotor element. The NAB and some of the ORF transcripts initiate 1 nucleotide before the coding region; the L11e monocistronic transcript initiates precisely at the first A of the initiator methionine ATG codon. The Lie - L10e - L12e tricistronic transcript has a 75 nucleotide leader that is probably involved in the autogenous regulation of the transcript at the translational level by the Lie protein. Termination of transcription occurs, with a single exception, within T tracts after GC rich regions. Although classic Shine-Dalgarno (eubacterial) type ribosome binding sites are present upstream of the Lie and L10e genes, the mechanism of translation initiation for transcripts with nil or negligible 5' leaders remains to be elucidated. Alignments between the deduced amino acid sequences of the L1le, Lie, Ll0e and L12eribosomal proteins and other available homologous proteins of archaebacteria, eubacteria and eucaryota have been made and show that the L11e, Lie and L10e proteins are colinear whereas the L12e protein has suffered a rearrangement through what appears to be gene fusion events. The L11e proteins exhibit (i) sequence conservation in the region interacting with release factor 1, (ii) conserved proline residues (probably contributing to the elongated shape of the molecule) and (iii) sites of methylation in Eco L11 are not conserved in the archaebacterial L11e proteins. The Lie proteins have regions of very high sequence similarity near the center and carboxy termini of the proteins but the relationships between protein structure and function remain unknown. Intraspecies comparisons between L10e and L12e sequences indicate the archaebacterial and eucaryotic L10e proteins contain a partial copy of the L12e protein fused to their carboxy terminus. In the eubacteria most of this fusion has been removed by a carboxy terminal deletion. Within the L12e derived region a 26 amino acid long internal modular sequence reiterated thrice in the archaebacterial L10e, twice in the eucaryotic L10e and once in the eubacterial L10e was discovered. This modular sequence also appears to be present in single copy in all Ll2e proteins and may play a role in L12e dimerization, L10e - L12e complex formation and the function of L10e - L12e complex in translation. From these sequence comparisons a model depicting the evolutionary progression gene cluster and proteins from the primordial state to the contemporary archaebacterial, eucaryotic and eubacterial states is presented. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Ribosome synthesis is a very complex and energy consuming process in which pre-ribosomal RNA (pre-rRNA) processing and folding events, sequential binding of ribosomal proteins and the input of approximately 200 trans-acting ribosome assembly factors need to be tightly coordinated. In the yeast Saccharomyces cerevisiae, ribosome assembly starts in the nucleolus with the formation of a very large 90S-sized complex. This ~2.2MDa pre-ribosomal complex is subsequently processed into the 40S and 60S assembly intermediates (pre-40S and pre-60S), which subsequently mature largely independently. Although we have a fairly complete picture of the protein composition of these pre-ribosomes, still very little is known about the rRNA structural rearrangements that take place during the assembly of the 40S and 60S subunits and the role of the ribosome assembly factors in this process. To address this, the Granneman lab developed a method called ChemModSeq, which made it possible to generate nucleotide resolution maps of RNA flexibility in ribonucleoprotein complexes by combining SHAPE chemical probing, high-throughput sequencing and statistical modelling. By applying ChemModSeq to ribosome assembly intermediates, we were able to obtain nucleotide resolution insights into rRNA structural rearrangements during late (cytoplasmic) stages of 40S assembly and for the early (nucleolar) stages of 60S assembly. The results revealed structurally distinct cytoplasmic pre-40S particles in which rRNA restructuring events coincide with the hierarchical dissociation of assembly factors. These rearrangements are required to trigger stable incorporation of a number of ribosomal proteins and the completion of the head domain. Rps17, one of the ribosomal proteins that fully assembled into pre-40S complexes only at a later assembly stage, was further characterized. Surprisingly, my ChemModSeq analyses of nucleolar pre-60S complexes indicated that most of the rRNA folding steps take place at a very specific stage of maturation. One of the most striking observations was the stabilization of 5.8S pre-rRNA region, which coincided with the dissociation of the assembly factor Rrp5 and stable incorporation of a number of ribosomal proteins.

By determining the Calpha and Cbeta chemical shift of P2 and its relaxation properties, together with secondary structure prediction, P2 was found to have an N-terminal 4-helices structural domain and a C-terminal unstructured coil. / P2 was found to interact with P1, forming heterodimer and with P2, forming homodimer. It was found that dimerization is carried out by their N-terminal, forming NTD-P1/NTD-P2 heterodimer and NTD-P2 homodimer. / Ribosome is the organelle responsible for protein synthesis and it was suggested that P-proteins located at the lateral stalk are involved in this process. Until now, structure of any P-protein is still not known although crystal structure of ribosome was solved. In order to know more about the biological role of P-proteins, structural characterization was carried out on human ribosomal protein P2. / The C-terminal tail which is conserved among P0, P1 and P2 of various species was found to interact with ribosome inactivating protein (TCS). This helps delivering TCS to its RNA substrate and carrying out its N-glycosidase activity. It was also found that TCS and EF2 are close in space suggesting that binding of TCS to P-proteins may hinder the association of EF2 to P-protein, thus inhibiting protein translation. / The solution structure of NTD-P2 homodimer was solved. It has 8 helices, 4 from each monomer. The surface is hydrophilic and the core is hydrophobic with a hydrophobic dimeric interface. By circular dicroism measurement, structural alignment and secondary structure prediction, we hypothesize that the dimerization mode of NTD-P1/NTD-P2 heterocomplex should be similar to NTD-P2 homodimer. Therefore, homology modeling was used to model the structure of NTD-P1/NTD-P2 using NTD-P2 as template. Interestingly, there is a small exposed hydrophobic patch on NTD-P1 which is lack in NTD-P2. This exposed hydrophobic patch may be the potential P0 binding site, forming P0(P1/P2)2 complex. Moreover, this exposed hydrophobic pocket is smaller than that of prokaryotic counterpart, thus providing insight in ribosome assembly and regulation in protein translation. / Lee, Ka Ming. / Advisers: K. B. Wong; P. C. Shaw. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0096. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 121-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Diamond Blackfan Anemia is a rare congenital disease that is primarily characterized by reduced erythroid progenitors. DBA pathogenesis has been associated with genes encoding ribosomal proteins (RPs) which are important in translation. However, this fails to explain why erythropoiesis is specifically disrupted. Our lab previously found that aberrant splicing of the human transcript encoding heme exporter, FLVCR1, is involved in DBA pathogenesis; and that RPS19 implicated in 25% of DBA patients, regulates FLVCR1 transcript splicing. This thesis investigated the role of another DBA associated gene encoding RPS17, in the regulation of FLVCR1 splicing and disrupted erythropoiesis in DBA. My findings further support the role of FLVCR1 aberrant splicing in DBA and provide evidence suggesting that RPS17 may not be a candidate DBA gene. Furthermore, my study implicates a potential role for RPS19 transcript levels in defective erythroid differentiation observed in DBA.

Diamond Blackfan Anemia is a rare congenital disease that is primarily characterized by reduced erythroid progenitors. DBA pathogenesis has been associated with genes encoding ribosomal proteins (RPs) which are important in translation. However, this fails to explain why erythropoiesis is specifically disrupted. Our lab previously found that aberrant splicing of the human transcript encoding heme exporter, FLVCR1, is involved in DBA pathogenesis; and that RPS19 implicated in 25% of DBA patients, regulates FLVCR1 transcript splicing. This thesis investigated the role of another DBA associated gene encoding RPS17, in the regulation of FLVCR1 splicing and disrupted erythropoiesis in DBA. My findings further support the role of FLVCR1 aberrant splicing in DBA and provide evidence suggesting that RPS17 may not be a candidate DBA gene. Furthermore, my study implicates a potential role for RPS19 transcript levels in defective erythroid differentiation observed in DBA.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.