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

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From its introduction in 1952 onwards, the clinical use of macrolides has been steadily increasing, both in human and veterinary medicine. Although initially designed to the treatment of Grampositive microorganisms, this antimicrobial family has also been used to treat specific Gram-negative bacteria. Some of them, as azithromycin, are considered in the armamentarium against Enterobacteriaceae infections. However, the facility that this bacterial genus has to gain or develop mechanisms of antibiotic resistance may compromise the future usefulness of these antibiotics to fight against Enterobacteriaceae infections. The present review is focused on the mechanisms of macrolide resistance, currently described in Enterobacteriaceae.

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.

Frequent rearrangements, typically through homologous recombination in plant mitochondrial genomes often result in different upstream and downstream sequences for the same gene among a number of species. Transcription and RNA processing signals are therefore different, even among closely related plants. To evaluate the impact of DNA rearrangements on gene expression I conducted a comparative analysis of the S7 ribosomal protein gene (rps7) among a number of grasses: wheat, rice, maize, barley, rye, brome, Lolium and oats (grasses whose evolutionary divergence times range from about 5 to 60 Mya). Using circularized-RT-PCR to simultaneously map rps7 transcript termini I found that 3’ends for various RNA species are homogeneous, mapping to conserved sequences among plants. 5’ termini are more complex and can be both discrete and heterogeneous for different transcripts, both within and among plants. Genome rearrangements upstream of the rps7 start codon for some but not all species has led to plant-specific signals for both rps7 transcription and RNA processing. Termini for rps7 precursor species in wheat and Lolium are very discrete and likely use different upstream tRNAs as processing signals for end-cleavage. A number of potential stem-loop structures have also been identified at or near 5’ and 3’ termini which may function in maturation of transcript ends or provide transcript stability and protection from degradation by ribonucleases. C-to-U RNA editing of non-coding sequences, a rare event, was observed at multiple sites within the 5’ and 3’UTRs among plants. Some sites may even be developmentally regulated as CR-RT-PCR experiments were conducted using mitochondrial RNA isolated from seedlings and germinating embryos. Taken together, my observations demonstrate the frequency of upstream DNA rearrangements and the variety of signals used for expression of rps7 among grasses, providing new insights into the complexities of mRNA production in plant mitochondria.

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.