Thesis advisor: Steven D. Bruner / Peptidic natural products are produced by diverse organisms ranging from bacteria to humans. These secondary metabolites can be assembled by the ribosome or by nonribosomal peptide synthetase (NRPS) enzymatic assembly lines. The architectural complexity and biological activity of such compounds make them interesting targets for study. Frequently, nonribosomal peptides contain nonproteinogenic amino acid building blocks, and the biosynthetic routes to both ribosomal and nonribosomal peptides often utilize tailoring enzymes. These specialized enzymes catalyze mechanistically challenging reactions and provide peptidic natural products with structural motifs not normally found in proteins. Structural studies of these tailoring enzymes will further our understanding of biosynthetic pathways, and engineered tailoring enzymes could find use as promiscuous catalysts for the chemoenzymatic synthesis of natural product analogs. The L-tyrosine 2,3-aminomutase <italic>Sg</italic>TAM catalyzes the formation of &beta;-tyrosine from L-tyrosine, and is used in the biosynthetic pathway to the enediyne antitumor antibiotic C-1027. This enzyme contains the rare electrophilic prosthetic group 4-methylideneimidazole-5-one (MIO) and is homologous to the histidine ammonia lyase family of enzymes. While lyases form &alpha;,&beta;-unsaturated carboxylates as products, <italic>Sg</italic>TAM catalyzes additional chemical steps that result in an overall 2,3-amino shift. The precise mechanistic role of MIO in the ammonia lyase and aminomutase families of enzymes was actively debated for over 50 years. Here, we report the first x-ray crystal structure of an MIO-dependent aminomutase and detail the synthesis and characterization of mechanistic probes for this enzyme. Furthermore, we report several structures of <italic>Sg</italic>TAM bound to substrate analogs. These co-crystal structures reveal how <italic>Sg</italic>TAM achieves substrate recognition and suggest a specific role for MIO in catalysis. The results of our studies allow for the rational engineering of MIO-based aminomutases and ammonia lyases with altered physical properties and substrate specificities. Additionally, we are currently studying several enzymes involved in the biosynthesis of the tricyclic depsipeptide microviridin J. This ribosomal peptide natural product contains two lactones and one lactam, which are introduced by two enzymes belonging to the ATP-grasp ligase superfamily of proteins. Here, we detail the overexpression of these enzymes, MdnJ-B and MdnJ-C, in <italic>E. coli</italic> and report the optimization of conditions which lead to the crystallization of both enzymes. The structural characterization of MdnJ-B and MdnJ-C will lead to a greater understanding of macrocycle formation in ribosomal peptide biosynthesis, and engineered variants of these enzymes may find use as macrocylcization catalysts. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Cheng, Tat Cheung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 181-188). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Leung Tak Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 57-63). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / Abbreviations --- p.iii / Abbreviations of amino acids --- p.iv / Abbreviations of nucleotides --- p.iv / Naming system for TRP mutants --- p.v / Chapter Chapter 1 --- I ntroduction / Chapter 1.1 --- Hyperthermophile and hyperthermophilic proteins --- p.1 / Chapter 1.2 --- Hyperthermophilic proteina are highly similar to their mesophilic homologues --- p.2 / Chapter 1.3 --- Hyperthermophilic proteins and free energy of stabilization --- p.3 / Chapter 1.4 --- Mechanisms of protein stabilization --- p.4 / Chapter 1.5 --- The difference in protein stability between mesophilic protein and hyperthermophilic protein --- p.4 / Chapter 1.6 --- Ribosomal protein L30e from T. celer can be used as a model system to study thermostability --- p.9 / Chapter 1.7 --- Protein engineering of TRP --- p.10 / Chapter 1.8 --- Purpose of the present study --- p.12 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Bacterial strains --- p.13 / Chapter 2.2 --- Plasmids --- p.13 / Chapter 2.3 --- Bacterial culture media and solutions --- p.13 / Chapter 2.4 --- Antibiotic solutions --- p.13 / Chapter 2.5 --- Restriction endonucleases and other enzymes --- p.14 / Chapter 2.6 --- M9ZB medium --- p.14 / Chapter 2.7 --- SDS-PAGE --- p.14 / Chapter 2.8 --- Alkaline phosphatase buffer --- p.15 / Chapter 2.9 --- DNA agarose gel --- p.15 / Chapter 2.10 --- "Gel loading buffer, DNA" --- p.16 / Chapter 2.11 --- "Ethidium bromide (EtBr), lOmg/ml" --- p.16 / Chapter 2.12 --- Constructing mutant TRP genes --- p.16 / Chapter 2.12.1 --- Polymerase Chain Reaction (PCR) --- p.17 / Chapter 2.12.2 --- Gel electrophoresis --- p.19 / Chapter 2.12.3 --- DNA purification from agarose gel --- p.19 / Chapter 2.12.4 --- "Construction of R39A, R39M, K46A, K46M, E47A, E50A, R54A, R54M" --- p.19 / Chapter 2.12.5 --- "Construction of double mutant R39A/E62A, R39M/E62A" --- p.20 / Chapter 2.13 --- Sub-cloning --- p.21 / Chapter 2.13.1 --- Restriction digestion --- p.22 / Chapter 2.13.2 --- Ligation vector with mutant TRP gene insert --- p.22 / Chapter 2.13.3 --- Amplifying vector carrying mutant TRP gene insert --- p.22 / Chapter 2.13.4 --- Mini-preparation of DNA --- p.22 / Chapter 2.13.5 --- Preparations of competent cells --- p.23 / Chapter 2.13.6 --- Transformation of Escherichia coli --- p.24 / Chapter 2.13.7 --- Screening tests --- p.25 / Chapter 2.14 --- Over expression of mutant TRP --- p.26 / Chapter 2.14.1 --- Transformation --- p.26 / Chapter 2.14.2 --- Expression --- p.26 / Chapter 2.14.3 --- Cell harvesting --- p.27 / Chapter 2.14.4 --- Expression checking --- p.27 / Chapter 2.14.5 --- SDS-PAGE --- p.27 / Chapter 2.14.6 --- Staining the acrylamide gel --- p.28 / Chapter 2.15 --- Purification of mutant TRP protein --- p.28 / Chapter 2.15.1 --- Cells lysis --- p.28 / Chapter 2.15.2 --- Chromatography --- p.29 / Chapter 2.15.3 --- Concentrating TRP as protein stock --- p.31 / Chapter 2.16 --- Protein stability --- p.32 / Chapter 2.16.1 --- Chemical stability --- p.33 / Chapter 2.16.2 --- Thermal stability --- p.34 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Construction of mutant TRP genes --- p.36 / Chapter 3.1.1 --- PCR mutagenesis --- p.36 / Chapter 3.1.2 --- Sub-cloning of mutant TRP gene to express vector pET8c --- p.37 / Chapter 3.2 --- Expression and purification of mutant TRP --- p.38 / Chapter 3.3 --- Protein stability --- p.39 / Chapter 3.3.1 --- Free energy of unfolding --- p.39 / Chapter 3.3.2 --- Thermal stability --- p.43 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- "Effect of R39, K46, E62, E64" --- p.47 / Chapter 4.2 --- Double mutation at R39 and E62 --- p.50 / Chapter 4.3 --- Effect of R54 --- p.51 / Chapter 4.4 --- Effect of E47 and E50 --- p.53 / Chapter 4.5 --- Conclusion --- p.54 / References --- p.57 / Appendix --- p.64

by Patrick, Tik-wan Law. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 144-155). / Acknowledgements --- p.i / Contents --- p.ii / Abstract --- p.vi / Abbreviations --- p.viii / List of figures --- p.ix / List of tables --- p.xiv / Chapter Chapter One: --- Introduction --- p.1-17 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- The Human genome project --- p.2 / Chapter 1.3 --- The EST approach --- p.3 / Chapter 1.4 --- Significance of the EST approach --- p.3 / Chapter 1.5 --- Human heart cDNA sequencing --- p.5 / Chapter 1.6 --- Significance of the human adult heart EST project --- p.7 / Chapter 1.7 --- Ribosomal proteins --- p.8 / Chapter 1.7.1 --- The ribosomal constituents --- p.8 / Chapter 1.7.2 --- Eukaiyotic ribosomal proteins --- p.10 / Chapter 1.8 --- Mammalian ribosomal proteins --- p.11 / Chapter 1.8.1 --- Evolution of mammalian ribosomal proteins --- p.11 / Chapter 1.8.2 --- Significance of mammalian ribosomal proteins --- p.12 / Chapter 1.9 --- Possible functional roles of ribosomal protein --- p.14 / Chapter 1.10 --- Nomenclature of ribosomal proteins --- p.16 / Chapter 1.11 --- The theme of the thesis --- p.17 / Chapter Chapter Two: --- Materials and Methods --- p.18-49 / Chapter 2.1 --- Cycle sequencing --- p.18 / Chapter 2.1.1 --- Plating out the cDNA library --- p.18 / Chapter 2.1.2 --- Amplification of the cDNA clones by PCR --- p.19 / Chapter 2.1.3 --- Purification and quantitation of the PCR product --- p.20 / Chapter 2.1.4 --- Cycle DNA sequencing --- p.20 / Chapter 2.2 --- Cloning of hrpL29 in pUC 18 cloning vector --- p.21 / Chapter 2.2.1 --- Amplification of the phage by plate lysate --- p.21 / Chapter 2.2.2 --- Amplification of the insert by PCR --- p.22 / Chapter 2.3 --- Screening for hrpL29 transformant --- p.22 / Chapter 2.3.1 --- Mini-preparation of plasmid DNA (Sambrook et al，1989) --- p.22 / Chapter 2.3.2 --- Large scale preparation of plasmid DNA --- p.24 / Chapter 2.4 --- Primer design for cloning of an intron of hrpL29 --- p.26 / Chapter 2.5 --- Isolation of the intron of hrpL29 by PCR --- p.26 / Chapter 2.6 --- Restricted endonuclease digestion --- p.27 / Chapter 2.7 --- Purification of DNA from the agarose gel --- p.27 / Chapter 2.8 --- Dephosphorylation of linearized plasmid DNA --- p.29 / Chapter 2.9 --- DNA ligation --- p.29 / Chapter 2.10 --- "Preparation of competent bacterial cells for transformation (Hanahan,1985)" --- p.30 / Chapter 2.11 --- Plasmid DNA Transformation --- p.31 / Chapter 2.12 --- Unicycle DNA sequencing by T7 polymerase (Pharmacia) --- p.32 / Chapter 2.13 --- Synthesis of radiolabelled DNA probe --- p.33 / Chapter 2.14 --- "Oligonucleotide synthesis, deprotection and purification" --- p.34 / Chapter 2.14.1 --- Oligonucleotide synthesis --- p.34 / Chapter 2.14.2 --- Deprotection and purification of oligonucleotides --- p.35 / Chapter 2.15 --- Southern analysis --- p.36 / Chapter 2.15.1 --- "Isolation of genomic DNA from leukocytes (Ciulla et al,1988)" --- p.36 / Chapter 2.15.2 --- Restricted digestion and fractionation of genomic DNA --- p.37 / Chapter 2.15.3 --- Southern transfer of DNA onto a membrane support --- p.37 / Chapter 2.15.4 --- Prehybridization of the Southern blot --- p.40 / Chapter 2.15.5 --- Hybridization of the Southern blot --- p.40 / Chapter 2.16 --- Northern analysis --- p.41 / Chapter 2.16.1 --- "Isolation of total RNA by using the AGPC-RNA method (Chomczynski and Sacchi,1987, modified)" --- p.41 / Chapter 2.16.2 --- Separation of total RNA by electrophoresis and transfer onto a membrane support --- p.43 / Chapter 2.16.3 --- Prehybridization of the Northern blot --- p.46 / Chapter 2.16.4 --- Hybridization of the Northern blot --- p.47 / Chapter 2.17 --- First strand cDNA synthesis (Pharmacia) --- p.48 / Chapter 2.18 --- PCR of the first strand cDNA --- p.48 / Chapter Chapter Three: --- Results --- p.50-113 / Chapter 3.1 --- Partial sequencing of adult human heart cDNA clones --- p.50 / Chapter 3.2 --- DNA homology searching by using the program BLASTN --- p.52 / Chapter 3.2.1 --- Catalogue of the 502 ESTs of the cardiovascular system --- p.54 / Chapter 3.2.2 --- Classification and frequency of the human adult heart cDNA clones --- p.63 / Chapter 3.3 --- Submission of the cDNA sequences to NCBI --- p.64 / Chapter 3.4 --- Pattern of gene expression in the human adult cardiovascular system --- p.66 / Chapter 3.5 --- "Sequence determination of hrpL29 (Law et. al., 1996)" --- p.72 / Chapter 3.5.1 --- Cycle Taq sequencing of hrpL29 --- p.72 / Chapter 3.5.2 --- Subcloning of the hrpL29 cDNA insert into the pUC18 DNA cloning vector --- p.75 / Chapter 3.5.3 --- Unicycle T7 sequencing of hrpL29 --- p.77 / Chapter 3.6 --- Sequence alignment and comparison of hrpL29 with other known sequences in the databases --- p.79 / Chapter 3.7 --- The primary structure of hrpL29 --- p.83 / Chapter 3.8 --- Results of RT-PCR and PCR --- p.88 / Chapter 3.9 --- Genomic analysis of hrpL29 --- p.92 / Chapter 3.9.1 --- Isolation of the first intron of hrpL29 --- p.92 / Chapter 3.9.2 --- Southern analysis of hrpL29 --- p.97 / Chapter 3.10 --- Northern analysis of hrpL29 --- p.103 / Chapter 3.10.1 --- Tissue distribution of hrpL29 mRNA in rat tissues --- p.103 / Chapter 3.10.2 --- Time course of hRPL29 expression in mouse heart --- p.106 / Chapter 3.10.3 --- Time course of hRPL29 expression in mouse brain --- p.110 / Chapter Chapter Four: --- Discussion --- p.114-139 / Chapter 4.1 --- Characterization of the ESTs --- p.114 / Chapter 4.2 --- Significance of the heart EST project --- p.116 / Chapter 4.3 --- Redundancy of the EST sequencing --- p.118 / Chapter 4.4 --- The importance of frequent database searching --- p.119 / Chapter 4.5 --- The importance of an efficient comparison algorithm --- p.120 / Chapter 4.6 --- Human ribosomal protein L29 (hRPL29) --- p.122 / Chapter 4.7 --- Internal duplication in hRPL29 --- p.124 / Chapter 4.8 --- Primary structure analysis of hRPL29 --- p.126 / Chapter 4.9 --- RT-PCR and PCR of the first strand cDNA with primers using the C095-ATG and dT primer --- p.128 / Chapter 4.10 --- Southern analysis of hrpL29 --- p.128 / Chapter 4.11 --- Northern analysis of hrpL29 --- p.133 / Chapter 4.11.1 --- Tissue distribution of the mRNA species of hrpL29 --- p.133 / Chapter 4.11.2 --- Time course of hRPL29 expression in mouse heart and brain --- p.134 / Chapter 4.12 --- Possible functional role of hRPL29 --- p.135 / Chapter 4.13 --- Further aspects --- p.137 / Appendix --- p.140-143 / References --- p.144-155

The nucleolus is the site for rRNA synthesis, a process requiring the recruitment of many proteins involved in ribosomal biogenesis. Nrap is a novel nucleolar protein found to be present in all eukaryotes. Preliminary characterisation of Nrap suggested it was likely to participate in ribosome biogenesis but as with many other nucleolar proteins, the functional role of Nrap is largely unknown. In this study, the role of mammalian Nrap in the nucleolus and in ribosome biogenesis was explored. Initially, a number of tools were generated to investigate Nrap function. This involved raising and purifying a polyclonal antibody against the N-terminal region of Nrap. The anti-Nrap antibody was found to detect two Nrap bands in mouse fibroblast cells, possibly corresponding to the two mouse Nrap isoforms, and . In addition, mammalian expression vectors containing the full Nrap sequence as well as deletion constructs were created. The subcellular localisation of each construct was observed by fluorescent microscopy. It was revealed that recombinant Nrap did not localise to the nucleolus, possibly because it was exported to undergo degradation by the 26S proteasome. Two putative NLSs were found to be responsible for directing Nrap to the nucleus but a region accountable for nucleolar localisation was not identified. The data indicated that multiple domains working together are likely to direct Nrap to the nucleolus. Nrap was also observed to co-localise with nucleolar proteins B23 and p19ARF. Moreover, it was shown by reciprocal immunoprecipitation that these three nucleolar proteins existed in a complex in unsynchronised mouse fibroblast cells. Recent reports demonstrated a complex relationship between B23 and p19ARF although the functional significance remained unclear. Nrap's in vivo association with B23 and p19ARF indicated a specific functional role in the nucleolus. Nrap knockdown using siRNA significantly increased B23 protein levels in a dose-dependent manner and down-regulated p19ARF protein levels at higher siRNA concentration. Preliminary studies also implicated Nrap in cell proliferation through these novel interactions. Both endogenous and recombinant Nrap were found to be highly unstable suggesting that Nrap might regulate B23 and p19ARF through its own tightly regulated stability. Finally, the role of Nrap in rRNA processing was investigated by northern blot analysis. Nrap knockdown was found to affect the levels of 45S, 32S and 28S rRNAs. The changes found may be a consequence of the concurrent perturbation in the levels of B23 and p19ARF caused by Nrap knockdown. As the results were not consistent with previous reports, it was likely that changes to rRNA processing could be contributed to Nrap loss of function. This study demonstrated for the first time a functional role of Nrap in rRNA processing possibly through its association with B23 and p19ARF.

<p>Peptide bond formation and translational termination on the ribosome have been simulated by molecular mechanics, free energy perturbation, empirical valence bond (MD/FEP/EVB) and automated docking methods. Recent X-ray crystallographic data is used here to calculate the entire free energy surface for the system complete with substrates, ribosomal groups, solvent molecules and ions. A reaction mechanism for peptide bond formation emerges that is found to be catalyzed by the ribosome, in agreement with kinetic data and activation entropy measurements. The results show a water mediated network of hydrogen bonds, capable of reducing the reorganization energy during peptidyl transfer. The predicted hydrogen bonds and the structure of the active site were later confirmed by new X-ray structures with proper transition states analogs. </p><p>Elongation termination on the ribosome is triggered by binding of a release factor (RF) protein followed by rapid release of the nascent peptide. The structure of the RF, bound to the ribosomal peptidyl transfer center (PTC), has not been resolved in atomic detail. Nor is the mechanism known, by which the hydrolysis proceeds. Using automated docking of a hepta-peptide RF fragment, containing the highly conserved GGQ motif, we identified a conformation capable of catalyzing peptide hydrolysis. The MD/FEP/EVB calculations also reproduce the slow spontaneous release when RF is absent, and rationalize available mutational data. The network of hydrogen bonds, the active site structure, and the reaction mechanism are found to be very similar for both peptidyl transfer and termination. </p><p>New structural data, placing a ribosomal protein (L27) in the PTC, motivated additional MD/FEP/EVB simulations to determine the effect of this protein on peptidyl transfer. The simulations predict that the protein N terminus interacts with the A-site substrate in a way that promotes binding. The catalytic effect of L27 in the ribosome, however, is shown to be marginal and it therefore seems valid to view the PTC as a ribozyme. Simulations with the model substrate puromycin (Pmn) predicts that protonation of the N terminus can reduce the rate of peptidyl transfer. This could explain the different pH-rate profiles measured for Pmn, compared to other substrates.</p>

Peptide bond formation and translational termination on the ribosome have been simulated by molecular mechanics, free energy perturbation, empirical valence bond (MD/FEP/EVB) and automated docking methods. Recent X-ray crystallographic data is used here to calculate the entire free energy surface for the system complete with substrates, ribosomal groups, solvent molecules and ions. A reaction mechanism for peptide bond formation emerges that is found to be catalyzed by the ribosome, in agreement with kinetic data and activation entropy measurements. The results show a water mediated network of hydrogen bonds, capable of reducing the reorganization energy during peptidyl transfer. The predicted hydrogen bonds and the structure of the active site were later confirmed by new X-ray structures with proper transition states analogs. Elongation termination on the ribosome is triggered by binding of a release factor (RF) protein followed by rapid release of the nascent peptide. The structure of the RF, bound to the ribosomal peptidyl transfer center (PTC), has not been resolved in atomic detail. Nor is the mechanism known, by which the hydrolysis proceeds. Using automated docking of a hepta-peptide RF fragment, containing the highly conserved GGQ motif, we identified a conformation capable of catalyzing peptide hydrolysis. The MD/FEP/EVB calculations also reproduce the slow spontaneous release when RF is absent, and rationalize available mutational data. The network of hydrogen bonds, the active site structure, and the reaction mechanism are found to be very similar for both peptidyl transfer and termination. New structural data, placing a ribosomal protein (L27) in the PTC, motivated additional MD/FEP/EVB simulations to determine the effect of this protein on peptidyl transfer. The simulations predict that the protein N terminus interacts with the A-site substrate in a way that promotes binding. The catalytic effect of L27 in the ribosome, however, is shown to be marginal and it therefore seems valid to view the PTC as a ribozyme. Simulations with the model substrate puromycin (Pmn) predicts that protonation of the N terminus can reduce the rate of peptidyl transfer. This could explain the different pH-rate profiles measured for Pmn, compared to other substrates.

The Sigma-54 Promoter Database contains computationally predicted sigma-54 promoters from over 60 prokaryotic species. Organisms from all major phyla were analysed and results were made available online at http://www.sigma54.ca. This database is particularly unique due to its inclusion of intragenic regions, grouping of data by COG and COG category, and the ability to summarize results either by phylum or database-wide. ClusterMine360 (http://www.clustermine360.ca/) is a database of microbial polyketide and nonribosomal peptide gene clusters. It takes advantage of crowd-sourcing by allowing members of the community to make contributions while automation is used to help achieve high data consistency and quality. The database currently has over 200 gene clusters from over 185 compound families. It also features a unique sequence repository containing over 10,000 PKS/NRPS domains. The sequences are filterable and downloadable as individual or multiple sequence FASTA files. This database will be a useful resource for members of the PKS/NRPS research community enabling them to keep up with the growing number of sequenced gene clusters and rapidly mine these clusters for functional information.

Eukaryotic cytosolic ribosomes are composed of two distinct subunits consisting of four individual ribosomal RNAs and, in Arabidopsis thaliana, 81 ribosomal proteins. Functional subunit assembly is dependent on the production of each ribosomal component. Arabidopsis thaliana r-protein genes exist in multi-gene families ranging in size from two to seven transcriptionally active members. The cytosolic RPS15a gene family consists of four members (RPS15aA, -C, -D and -F) that, at the amino acid level, share 87-100% identity. Using semi-quantitative RT-PCR I have shown that RPS15aC is not expressed and that transcript abundance differs both spatially and temporally among the remaining RPS15a genes in non-treated Arabidopsis tissues and in seedlings following a variety of abiotic stresses. A comprehensive analysis of the RPS15a 5' regulatory regions (RRs) using a series of deletion constructs was used to determine the minimal region required for gene expression and identify putative cis-regulatory elements. Transcription start site mapping using 5' RACE indicated multiple sites of initiation for RPS15aA and -F and only a single site for RPS15aD while all three genes contain a leader intron upstream of the start codon. Analysis of reporter gene activity in transgenic Arabidopsis containing a series of 5' RR deletion::GUS fusions showed that, similar to previous RT-PCR results, there was a trend for mitotically active tissues to stain for GUS activity. Putative cis-elements including the TELO box, PCNA Site II motif and pollen specific elements were identified. However, there was not always a clear correlation between the presence of a putative element and RPS15a transcript abundance or GUS activity. Although variation in transcriptional activity of each RPS15a gene has been observed, subcellular localization of both RPS15aA and -D in the nucleolus has been confirmed in planta by confocal microscopy. The results of this thesis research suggest while all three active RPS15a genes are transcriptionally regulated, additional post-transcriptional and/or translational regulation may be responsible for final RPS15a levels while differential isoform incorporation into ribosomal subunits may be the final point of r-protein regulation.

Ribosomal proteins (RPs), synthesized in the cytoplasm, need to be transported from the cytoplasm to the nucleolus (a nuclear compartment), where a single molecule of each RP assembles with rRNAs to form the large and small ribosomal subunits. The objectives of this research were to identify nuclear/nucleolar localization signals (NLSs/NoLSs; generally basic motifs) that mediate the transport of Arabidopsis RPL23aA, RPL15A and RPS8A into the nucleus and nucleolus, and to study transcriptional regulation and subcellular localization of RPs. While all previous research has shown that nucleolar localization of proteins is mediated by specific basic motifs, in this study, I showed that a specific number of basic motifs mediated nucleolar localization of RPL23aA, rather than any specific motifs. In this protein, single mutations of any of its eight putative NLSs (pNLSs) had no effect on nucleolar localization, however, the simultaneous mutation of all eight completely disrupted nucleolar localization, but had no effect on nuclear localization. Furthermore, mutation of any four of these pNLSs had no effect on localization, while mutation of more than four increasingly disrupted nucleolar localization, suggesting that any combination of four of the eight pNLSs is able to mediate nucleolar localization. These results support a charge-based system for the nucleolar localization of RPL23aA. While none of the eight pNLSs of RPL23aA were required for nuclear localization, in RPS8A and RPL15A, of the 10 pNLSs in each, the N-terminal two and three NLSs, respectively, were absolutely required for nuclear/nucleolar localization. Considering the presence of only a single molecule of each RP in any given ribosome, which obligates the presence of each RP in the nucleolus in equal quantities, I studied transcriptional regulation of Arabidopsis RP genes and the subcellular localization of five RP families to determine the extent of coordinated regulation of these processes. Variation of up to 300-fold was observed in the expression levels of RP genes. However, this variation was drastically reduced when the expression level was considered at the RP gene family level, indicating that coordinate regulation of expression of RP genes, coding for individual RP isoforms, is more stringent at the family level. Subcellular localization also showed differential targeting of RPs to the cytoplasm, nucleus and nucleolus, together with a significant difference in the nucleolar import rates of RPS8A and RPL15A. Although one could expect coordinated regulation of the processes preceding ribosomal subunit assembly in the nucleolus, my results suggest differential regulation of these processes.