>Magister Scientiae - MSc / Pharmaceutical research and development strategies rely on the constant discovery of novel natural products as potential drugs. Recent studies have shown that the microorganisms associated with sponges are the true producers of some previously isolated compounds. This study created a large collection of bacterial symbionts associated with the South African marine sponge, Hamacantha esperioides. The bioactivity assays performed, showed that 44 isolates produced compounds with antimicrobial or anti-inflammatory activity. The successful identification of novel species that produce potential natural products highlights the importance of cultivation-dependent methods. To further screen for natural products, a cultivation-independent approach was used. A sequenced-based method, based on the biosynthetic genes of polytheonamide, was developed to screen for proteusins in sponge metagenomic DNA and the genomes of bacterial symbionts. The degenerate primers could amplify the targeted genes from DNA known to contain homologues. Evaluation of the primers' specificity showed non-specific amplification of genes, some containing similar conserved domains as the target genes. This study demonstrated that the use and development of cultivation-dependent and -independent screens are important for the discovery of novel natural products from the symbiotic bacteria of South African sponges. / National Research Foundation (NRF)

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.

Massive mitochondrial gene transfer to the nucleus occurred very early during eukaryotic evolution following endosymbiosis, and is still ongoing in plants. Tracking recent gene transfer events can give us insight into the evolutionary processes by which a transferred gene becomes functional in the nucleus and how its protein gets targeted back into the mitochondrion, where it is needed. Rps19 and rpl2 are two such ribosomal protein genes that are known to have been transferred to the nucleus, many times independently during flowering plant evolution. My research project focusses on determining the status and expression of rps19 and rpl2 in the mitochondrion and nucleus of selected grasses and in particular brome (close relative to agronomically important crops such as wheat, rye and barley). My results at the level of DNA and RNA (PCR and RT-PCR, respectively) show that the mitochondrial brome rpl2 copy is a pseudogene while its functional gene is in the nucleus. The brome mitochondrial genome has a copy of rps19 which is transcribed and C-U edited. Surprisingly, the brome nuclear genome also has functional copies of rps19.The targeting sequence for the nuclear rps19 gene was acquired from duplication of mitochondrial targeting heat shock protein (hsp70) presequence. Comparative analysis strongly suggests that a functional rps19 gene was transferred to the nucleus before rice and maize lineages split and now that brome rps19 has been found to be present in both compartments, this implies a transition stage of about 60 million years. Oats was found to have a functional rps19 copy in the nucleus and has a novel presequence due to lineage specific rearrangements and exon shuffling. Functional paralogous copies were found in wheat, and maize while barley lost one of the copy. Thus, following transfer, duplication of rps19 gene must have occurred in the ancestor of barley and wheat clade. Maize might have had a recent duplication or gene conversion events along its lineage as its paralogous copies are very similar to each other. More information is needed to determine if this duplication event extends to wheat-brome, wheat-oats or even before rice and maize split. Barley was also found to have a recent independent DNA mediated transfer in addition to the common transfer, as it possesses an unedited nuc-mt rps19 in its nuclear genome. This suggests that barley must also have had a transition stage for ~60MY and lost its mitochondrial copy very recently.

The work presented explored the in vivo and in vitro synthesis of ribosomal RNA in the Gram positive, spore-forming bacterium Bacillus subtilis. The investigation began with a study of rRNA synthesis in B. subtilis during steady state growth and under nutritional shift-up conditions. The percent of transcription which is ribosomal RNA was measured by hybridization of pulse labeled RNA to a specific DNA probe carrying the 3' end of the 23S RNA gene. The fractional rate of ribosomal RNA synthesis increased with cellular growth rate, and showed a rapid increase after a nutritional shift up. RNA synthesis during infection with an amber mutant of bacteriophage SP01 was also examined. Infected cells continued to synthesize rRNA at the preinfection rate, but could not respond to media enrichment by increasing the percent rRNA-synthesis. The latter study suggested the existence of a specific RNA polymerase that transcribed ribosomal RNA genes. The conclusions from the in vivo study led to an analysis of rRNA transcription in vitro. The isolation of the putative ribosomal RNA specific RNA polymerase was attempted by affinity chromatography on cellulose complexed with plasmid DNA containing the promoter region of the B. subtilis rrnB rRNA operon, and by sedimentation through a glycerol gradient. No difference in activity profile was observed when transcription activity at the rRNA tandem promoters was compared to activity at a non-ribosomal promoter. Since in vivo analysis of the control of rRNA synthesis in Escherichia coli suggested that regulation occurs at the level of transcription initiation, in vitro transcription initiation at the B. subtilis rRNA promoters was investigated using the single round transcription assay. Initial rates of transcription were different at each of the two tandem promoters of the B. subtilis rrnB operon: the upstream promoter, PI, initiated slowly, while the downstream promoter, P2, initiated faster. In addition, transcription initiation at the two promoters appeared to be linked. The formation of a heparin resistant complex at the PI promoter affected the stability of the heparin resistant complex formed at the P2 promoter. The kinetics of transcription initiation at the tandem rRNA promoters were examined using the tau plot analysis. RNA polymerase had a high affinity for both rRNA promoters, but the rate of initiation at these promoters was relatively slow when compared to non-ribosomal promoters. Finally, transcription initiation on two artificial tandem promoter constructs was compared with initiation on the native tandem promoter construct. In general, PI was shown to have a positive effect on transcription from downstream promoters, but had specific effects on different promoters. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The work in this thesis addresses the general problem of how ribosomal subunits are exported from the nucleus to mature in the cytoplasm. There are three parts in this dissertation. In the first part, I asked questions about the specificity for export receptors in the nuclear export of the large (60S) ribosomal subunit in yeast. In principle, I tethered different export receptors that are known to work in various unrelated export pathways to the ribosome by fusing them to the trans-acting factor Nmd3. Interestingly, all the chimeric receptors were able to support export, although to different degrees. Moreover, 60S export driven by these chimeric receptors was independent of Crm1, an export receptor that is essential for 60S export in wild-type cells. The second question I addressed in this project was whether or not a nuclear export signal could be provided in cis on ribosomal proteins (Rpls) rather than in trans by a transacting factor. The nuclear export signal (NES) of Nmd3 was fused to different ribosomal proteins and tested for support of 60S export. Several Rpl-NES fusion constructs worked to promote 60S export. Rpl3 gave the best efficiency. In conclusion, these results imply unexpected flexibility in the 60S export pathway. This may help explain how different export receptors could have evolved in different eukaryotic lineages. In the second part of my thesis, I identified the assembly pathway for the base of the ribosome stalk. The stalk is an important functional domain of the large ribosomal subunit because of its requirement for interaction with translation factors. Mrt4 is a nuclear paralog of P0, which is an essential part of the stalk. Here, I identified Yvh1 a novel ribosome biogenesis factor that is required for the release of Mrt4. Yvh1 is a conserved dual phosphatase, but the C-terminal zinc-binding domain rather than the phosphatase function was required for its activity to release Mrt4. Mrt4 localizes in the nucleus and nucleolus in the wild-type cells, but was persistent on cytoplasmic 60S subunits in yvh1[Delta] cells. The persistence of Mrt4 on the 60S subunits blocked the loading of P0 and assembly of the stalk. I also found the binding of Yvh1 depended on Rpl12, a protein that binds together with P0 to form the base of the stalk. Deletion of Rpl12 phenocopied yvh1[Delta]. These data identified the function of Yvh1 as a release factor of Mrt4. I also showed that the function of Yvh1 is conserved in human cells. In my final project, I analyzed the interdependence and order of the known cytoplasmic maturation events of the 60S subunit. 60S subunits require several maturation steps in the cytoplasm before they become competent in translation. There are four major steps involving two ATPases, Drg1 and Ssa1, and two GTPases, Efl1 and Lsg1. In my study, I ordered these steps into one serial pathway. Drg1 releases Rlp24 in the earliest step of 60S maturation in the cytoplasm. Truncation of the C-terminus of Rlp24 blocked cytoplasmic maturation of the large subunit by preventing the recruitment of Drg1 and led to a secondary defect in the release of Arx1 because of a failure to recruit Rei1. Deletion of REI1 mislocalized Tif6 from the nucleus and nucleolus to the cytoplasm and deletion of ARX1 suppressed the Tif6 mislocalization, indicating that the release of Arx1 was required for Tif6 release downstream. I found that mutation of efl1 or sdo1, the known release factors for Tif6, also blocked Nmd3 release. Tif6-V192F, which could bypass the growth defects of efl1 or sdo1 mutants, suppressed the defect of Nmd3 recycling. These results showed that the release of Tif6 was a prerequisite for Nmd3 release. Thus, the release of Nmd3 is downstream of the Tif6 release step. In conclusion, I have ordered the events of cytoplasmic maturation with Drg1 as the first step after ribosome export, followed by Rei1/Jji1 and then Sdo1/Efl1. The release of Nmd3 by Lsg1 appears to be the last step of ribosome maturation in the cytoplasm. Thus, the two ATPases Drg1 and Ssa work first and then the two GTPases Efl1 and Lsg1 work in a linear pathway of 60S maturation in the cytoplasm. / text

Cell growth and proliferation are processes in the cell that must be tightly regulated. Transcription of ribosomal RNA and ribosomal biogenesis are directly linked to cell growth and proliferation, since the ribosomal RNA encodes for the majority of transcription in a cell and ribosomal biogenesis influences directly the number of proteins that are synthesized. In the work presented in this thesis, we have investigated the ribosomal RNA genes, namely the ribosomal DNA genes and the 5S rRNA genes, and their transcriptional regulation. One protein complex that is involved in RNA polymerase I and III transcription is the chromatin remodelling complex B‑WICH (WSTF, SNF2h, NM1). RNA polymerase I transcribes the rDNA gene, while RNA polymerase III transcribes the 5S rRNA gene, among others. In Study I we determined the mechanism by which B‑WICH is involved in regulating RNA polymerase I transcription. B‑WICH is associated with the rDNA gene and was able to create a more open chromatin structure, thereby facilitating the binding of HATs and the subsequent histone acetylation. This resulted in a more active transcription of the ribosomal DNA gene. In Study II we wanted to specify the role of NM1 in RNA polymerase I transcription. We found that NM1 is not capable of remodelling chromatin in the same way as B‑WICH, but we demonstrated also that NM1 is needed for active RNA polymerase I transcription and is able to attract the HAT PCAF. In Study III we investigated the intergenic part of the ribosomal DNA gene. We detected non-coding RNAs transcribed from the intergenic region that are transcribed by different RNA polymerases and that are regulated differently in different stress situations. Furthermore, these ncRNAs are distributed at different locations in the cell, suggesting that they have different functions. In Study IV we showed the involvement of B‑WICH in RNA Pol III transcription and, as we previously had shown in Study I, that B‑WICH is able to create a more open chromatin structure, in this case by acting as a licensing factor for c-Myc and the Myc/Max/Mxd network. Taken together, we have revealed the mechanism by which the B‑WICH complex is able to regulate RNA Pol I and Pol III transcription and we have determined the role of NM1 in the B‑WICH complex. We conclude that B‑WICH is an important factor in the regulation of cell growth and proliferation. Furthermore, we found that the intergenic spacer of the rDNA gene is actively transcribed, producing ncRNAs. Different cellular locations suggest that the ncRNAs have different functions. / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Manuscript; Paper 3: Manuscript</p>

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 4 uppsatser.

The bacterial ribosome is essential to cell growth yet little is known about how its proteins attain their mature structures. Recent studies indicate that certain Staphlyococcus aureus bacteriophage protein sequences contain specific sites that may be cleaved by a non-bacteriophage enzyme (Poliakov et al. 2008). The phage cleavage site was found to bear sequence similarity to the N-terminus of S. aureus ribosomal protein L27. Previous studies in E. coli (Wower et al.1998; Maguire et al. 2005) found that L27 is situated adjacent to the ribosomal peptidyl transferase site, where it likely aids in new peptide formation. The predicted S. aureus L27 protein contains an additional N-terminal sequence not observed within the N-terminus of the otherwise similar E. coli L27; this sequence appears to be cleaved, indicating yet-unobserved ribosomal protein post-translational processing and use of host processes by phage. Phylogenetic analysis shows that L27 processing has the potential to be highly conserved. Further study of this phenomenon may aid antibiotic development.

Thesis advisor: Marc Snapper / Thesis advisor: Steven Bruner / Peptide natural products have diverse, elaborate scaffolds and are important leads in the development of new drugs. A complete understanding of the natural biosynthetic pathways of these compounds can improve chemical syntheses and boost bioengineering efforts. There are two classes of peptide natural products: ribosomal and nonribosomal peptides. Ribosomally produced and posttranslationally modified peptides (RiPPs) are produced by the ribosome using the 20 canonical amino acids and undergo extensive tailoring to yield the active natural products. Nonribosomal peptides (NRPs) are assembled through an enzyme dependent system and can incorporate over 500 different amino and acyl building blocks to impart complexity. These peptides can also undergo additional tailoring to further modify the core peptide. The microviridins are a class of RiPPs that are modified by two ATP dependent ligases to create a total of three macrocyclic bonds. We have solved the three dimensional protein structures of each of these ligases to establish the mechanism of substrate recognition and cyclization. Vancomycin is a NRP that contains five nonproteinogenic aromatic amino acids that are necessary for biological activity. One of these amino acids is derived from a polyketide pathway and undergoes a four-electron oxidation by a cofactor independent dioxygenase, DpgC. We have solved the structure of this enzyme and have established a radical mechanism. We have investigated this mechanism using synthetic probes and mutagenesis. We have examined O₂ binding using molecular dynamics and mutagenesis. NRPs are synthesized by the multidomain, modular nonribosomal peptide synthetases (NRPSs) in an enzyme templated, ATP-dependent manner. We have synthesized domain specific probes to study the structures and mechanisms of these pathways. Our continued work will provide the insight necessary to manipulate these pathways to provide biologically active compounds. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Michelle M. Meyer / As the factory responsible for producing proteins, ribosomes are of great importance. In bacteria, ribosomes are composed of three ribosomal RNAs (rRNA) of different sizes, and around 50 ribosomal proteins (r-protein). During ribosome biogenesis in bacteria, synthesis of rRNAs and r-proteins are both tightly regulated and coordinated to ensure robust growth. In particular, a group of cis-regulatory RNA elements located in the 5' untranslated regions or the intergenic regions in r-protein operons are responsible for the regulation of r-protein biosynthesis. Based on the fact that RNA-regulated r-protein biosynthesis is essential and universal in bacteria, such unique and varied regulatory RNAs could provide new targets for antibacterial purpose. In this thesis, we report and experimentally verify a novel r-protein L1 regulation model that contains dual L1-binding RNA motif, and for the first time, a S6:S18 dimer-binding RNA structure in the S6 operon. We also describe Escherichia coli-based and Schizosaccharomyces pombe-based reporter systems for in vivo characterization of RNA-protein interactions. So far, both in vivo systems failed to report RNA-protein interactions, and thus need further tuning. In addition, we performed phage-display to select for regulatory RNA-binding small peptides and examined their effects on bacteria viability. One selected peptide, N-TVNFKLY-C, caused defective growth when overexpressed in E. coli. Yet, further studies must be conducted to verify the possibility that bacteria were killed by direct RNA-peptide interaction that disrupted the native r-protein regulation. / Thesis (MS) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.