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

Escherichia coli was grown in the presence of amino acid analogues or in the absence of required amino acids. The tRNAs. were isolated and characterized. Numerous changes were observed in the total tRNA acceptance for particular amino acids although in no instance did these changes occur for amino acids corresponding to the adverse growth condition. The isoacceptor patterns for particular labelled aminoacyl-tRNAs were determined on the anion exchanger RPC-5. Novel isoacceptor tRNAs were observed under several growth conditions. Significant changes in tRNA isoacceptor distributions were noted. In certain instances it appeared that changes in total amino acid acceptance could be explained in terms of the increased or decreased synthesis of particular tRNA isoacceptors while for other tRNAs it seemed that changes occurred in the synthesis of all isoacceptors for a particular amino acid such that the relative amounts of isoacceptors remained constant even when total amino acid acceptance changed considerably. E. coli was grown over a wide temperature range, 17°C to 44°C, and the tRNA isolated and characterized. Novel tRNA isoacceptors were observed at both high and low growth temperatures for most but not all tRNAs. It was shown that the same isoacceptors could be formed at both extremes of temperature. Preliminary results suggest that the novel isoacceptors are formed as the result of a temperature aggravation of a nutritional problem at extremes of growth temperature. One of the novel tRNA isoacceptors formed under a variety of adverse growth conditions, tRNA3[sup Val] , was purified and partially characterized. The results are consistent with tRNA3[sup Val] being an undermodified precursor of the major isoacceptor tRNA₁[sup Val]. E. coli str[sup D] was grown and the tRNA isolated and characterized. Major differences in the amino acid acceptances for several tRNAs were observed. These changes were accomplished without any significant changes in the relative isoacceptor distributions as determined by RPC-5 chromatography. Gel electrophoretic analysis was performed on tRNA from cells grown at extremes of growth temperature. Significant differences were observed in the 5S region; there was an accumulation of material in cells grown at low temperature and a decrease of material in cells grown at high temperature. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

No description available.

The 5' - termini of most eukaryotic and viral mRNAs contain the sequence of m7G(5')ppp(5')N- known as the "cap" structure. The guanine-7 methylation of this cap structure is important for mRNA processing and initiation of translation. A RNA (guanine-7)-methyltransferase that specifically methylates the 5'-terminal guanosine residue of the RNA cap structure has been purified about 280-fold from the nucleoplasm of Ehrlich ascites cells. This enzyme activity was elevated eight fold in Ehrlich ascites tumor cells when compared to the methyl transferase activity from normal mouse liver cells. The enzyme catalyzed the methyl group transfer from S-adenosylmethionine to a capped but unmethylated RNA substrate. The purified enzyme had an apparent native molecular weight of 95,000, as determined by gel filtration chromatography. SDS-PAGE showed a predominant protein band of 46,000 daltons suggesting that the native enzyme consisted of two identical subunits. The enzyme was unstable when the protein concentration was low but was stable when stored at -20°C in a buffer containing 50% glycerol. The (guanine-7)-methyltransferase showed a pH optimum at 8.0. Two monovalent ions, potassium and sodium, stimulate the enzyme activity with an optimum concentration of 0.05 M. The enzyme does not require magnesium for activity. Instead, this divalent ion and two others, manganese and calcium, were found to inhibit enzyme activity at concentrations as low as 5 mM. / Ph. D.

The segmented negative-sense vRNA genome of influenza A virus is replicated via a complementary RNA (cRNA) intermediate by the viral RNA-dependent RNA polymerase (RdRP). The replicative intermediate has been thought to exist as a complementary ribonucleoprotein (cRNP) complex. Development of an RNA-based affinity-purification strategy for the isolation of viral ribonucleoprotein complexes enabled the structural and functional characterisation of the previously uncharacterised cRNP complex. The cRNP exhibits a filamentous double-helical organisation with defined termini, containing the viral RdRP at one end and a loop structure at the other end. In vitro characterisation of cRNP activity led to a proposal of a model of vRNA synthesis that relies on a trans-activating RdRP. The viral RdRP is an important host range determinant indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Affinity-purification followed by mass spectrometry was performed to identify cellular proteins that interact with the viral RdRP. 171 cellular proteins were found to co-purify with the viral RdRP, the most abundant of which were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, mitochondrial and ribosomal proteins. Among the phosphatases, three subunits of the cellular serine/threonine-protein phosphatase 6 (PP6) were identified. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and siRNA-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. Taken together, these results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP.

Rift Valley fever (RVF) is a disease endemic to Africa, which has recently spread outside of Africa to the Arabian Peninsula. Rift Valley fever virus (RVFV) is the causative agent of RVF and manifests as severe hepatitis, encephalitis and haemorrhagic fever, resulting in mortality in approximately 1% of human cases. RVFV also affects agriculture as it causes high mortality rates in young ruminants (>90% in new-born lambs) and is associated with high levels of abortions, which results in devastating economic losses. RVFV is a single-stranded RNA virus with a genome comprising of three separate genetic elements referred to as the Large (L), Medium (M) and Small (S) segments. The negative sense L segment encodes an RNA-dependent RNA polymerase (RdRp) while the M segment encodes two glycoproteins, Gn and Gc, and two non-structural proteins, NSm1 and NSm2. The glycoproteins are important for viral entry, genome packaging and mature virion formation as well as being the main antigen for the elicitation of neutralising antibodies by humoral immunity. The NSm proteins are required for mosquito vector transmission and preventing viral-induced apoptosis in host cells. The ambisense S segment encodes in the positive orientation a non-structural (NSs) gene, and in the negative orientation the nucleocapsid (N) gene. NSs is an important virulence factor involved in subverting host defences and the loss of NSs results in a highly attenuated RVFV infection. N is required for RNA synthesis and encapsidation of viral genomes. There are currently very few treatments in the early stages of development and vaccines for RVFV are not readily available. The overall lack of therapeutic strategies for RVFV urges novel therapeutic development such as RNA interference (RNAi). Endogenous RNAi is triggered by dsRNA and is involved in gene regulation through sequence specific suppression of target mRNA. Therapeutic RNAi exploits the RNAi pathway to facilitate targeted degradation of viral genes and has been applied effectively to the inhibition of a number of viruses that cause chronic and acute infections. There are fewer studies that have used RNAi to inhibit highly pathogenic viruses. Efficacy has been demonstrated against Ebola virus, Lassa virus and Dengue fever virus, which suggests applicability to the inhibition of RVFV. In this thesis, short hairpin RNAs (shRNAs) were generated to target the NSs, N and M genes of RVFV, which are important proteins in the viral life cycle. To determine the knockdown efficacy of the shRNAs, HEK293 cells were transiently transfected with the shRNAs and a vector expressing the respective shRNA gene target fused to a luciferase reporter. The reporter levels were assessed using a dual-luciferase assay and several shRNAs were selected for further characterisation as a result of effective target knockdown. Consequently, the shRNAs reduced the levels of expressed FLAG-tagged NSs, N and M encoded proteins, which were detected using western blot analysis. ShRNAs directed against NSs were shown to disrupt this protein’s function to result in alleviation of pathogenic properties. Specifically, NSs was shown to suppress the transcription levels of a luciferase reporter as well as prevent the activation of an IFN-β promoter. When the shRNAs were transiently transfected into HEK293 cells, they were able to reverse NSs-induced suppression in the reporter assays. Furthermore, NSs is cytotoxic as determined by observing cell morphology under transmitted light microscopy, which was quantified using a MTT viability assay and cells that subsequently received anti-NSs shRNAs had improved viability. This class of anti-pathogenic shRNAs should be able to down-regulate NSs in vivo and attenuate RVFV virulence. However, NSs is not essential for viral replication and as a result of the aggressive pathology of haemorrhagic RVF, essential structural genes were targeted to investigate shRNAs with anti-replicative properties. ShRNAs directed against N were transfected 24 hrs prior to infection with RVFV. The inhibition of viral replication was determined by collecting supernatant over 3 days and measuring the levels of N antigen using an ELISA. The shRNAs demonstrated effective suppression of RVFV but N antigen was detected at 72 hrs post-infection, which suggested that the shRNAs were overwhelmed by the virus. A series of shRNAs against M were subsequently tested and the anti-M shRNAs effectively suppressed viral replication in cultured cells over an extended 96 hr experiment, demonstrating that M is a good target for RNAi-mediated inhibition of RVFV. In this thesis, the potential of RNAi-based therapeutics against RVFV was demonstrated and these data contribute to the growing knowledge that RNAi should be developed further as a potential treatment for haemorrhagic fever viruses. Finally, some DNA viruses such as HBV form cellular reservoirs from which new virus can be produced and the DNA is resistant to RNAi-mediated inhibition. RVFV is an RNA virus with an acute infection, which makes it more susceptible to RNAi and an excellent target for this particular therapeutic modality.

The influenza viral ribonucleoprotein complexes (vRNPs) are responsible for viral RNA synthesis. Each vRNP is comprised of one vRNA segment, the viral RNA dependent RNA polymerase complex (RdRP), and multiple copies of nucleoprotein (NP). NP serves as scaffold in formation of vRNPs, but also regulates vRNP activity. The N-terminus of NP contains a nonconventional nuclear localization signal (NLS1) essential for initial vRNP nuclear import, but also interacts with host RNA helicases to enhance viral RNA replication in the nucleus. NP contains at least one additional NLS sequence, with bioinformatics revealing a third NLS in some NP proteins. Published yeast-two hybrid results indicate that the first 20 amino acids of NP can sufficiently bind with cellular protein UAP56. Suggesting the interaction of NP-UAP56 can be a major mechanism of how NP involve in viral replication. Thus, to examine the role of the N-terminus of NP aside from its vRNP nuclear localization activity N-terminal 20 amino acid deletion mutants with or without the addition of the conventional NLS from SV-40 T-antigen were constructed, termed del20NLS-NP and del20-NP. Nuclear localization of vRNPs with these constructs was assessed by GFP expression and western blotting. All these constructs exhibit nuclear localization, consistent with NLS1 being utilized for vRNP localization but not NP localization and vRNP formation in the nucleus. Furthermore, qPCR results demonstrated decreased vRNA synthesis activity, exacerbated as the vRNA template is lengthened in both plasmids, consistent with a lack of interaction with host RNA helicases. Interestingly, del20-NP vRNP activity is less severe than del20NLS-NP, suggesting perturbations of the N-terminus disrupt vRNP activity. To narrow down the region responsible for vRNA expression defect, del10-NP was constructed. GFP expression displayed similar activity between del10-NP and WT-NP with del20-NP showing a severe defection, suggesting NP amino acids 11-20 might be the major region responsible for the vRNA synthesis defect. However, sucrose density gradient results do not support the published interaction between NP and UAP56 in 293T cells. These results support the N-terminal region, potentially amino acids 11-20 of NP, is playing the important role in efficient viral gene expression during virus replication especially as vRNA template lengthen, and that the NLS1 of NP is not essential for NP/vRNP nuclear localization in our reconstituted vRNP assay.

An extracellular bacteriocin-like substance produced by Clostridium acetobutylicum was detected during studies on an industrial fermentation process. The bacteriocin-like substance was not inducible by either ultraviolet light or mitomycin C, and its production was not associated with the induction of a protease. Studies on the mode of action of the bacteriocin-like substance indicated that it had no significant effect on DNA, RNA, or protein synthesis, and it did not cause the loss of intracellular ATP. However, the bacteriocin-like substance was able to lyse SDS-treated cells and cell walls of C. acetobutylicum and was identified as an autolysin. Some of the characteristics of this extracellular autolysin were determined, and after purification it was shown to be a glycoprotein with a molecular weight of 28 000.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

A síntese ribossomal é uma das maiores atividades em células eucarióticas. Este processo inicia-se no nucléolo e é finalizado após a exportação das subunidades 40S e 60S para o citoplasma. Três dos RNAs ribossomais de eucariotos (18S, 5.8S e 25S) são sintetizados como um transcrito primário de 35S, o qual é processado através de uma complexa e ordenada série de modificações nucleotídicas e clivagens endo e exonucleolíticas. Estas reações dependem de aproximadamente 170 proteínas, 80 small nucleolar RNAs e de seqüências no pré-rRNA. Os fatores trans-atuantes envolvidos no processamento podem ser agrupados como RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) e exonucleases, que incluem o complexo exossomo. O exossomo de levedura é formado por 10 proteínas essenciais que atuam na maturação de rRNAs, snRNAs, snoRNAs, além da degradação de mRNAs incorretamente processados. A estrutura do exossomo de archaea foi descrita recentemente, mas ainda não existem muitas informações sobre a regulação deste complexo e sobre a participação de cofatores que interagem de forma transiente com o exossomo. Diante disso, este trabalho visou a caracterização funcional das proteínas que formam o anel de RNases PH em Saccharomyces cerevisiae, assim como a caracterização estrutural e funcional de possíveis cofatores do exossomo de Saccharomyces cerevisiae, Nop17p e Ylr022p, e do exossomo de Pyrococcus, Pab418p, Pab1135p e aNip7p. Os dados obtidos evidenciam que a atividade exonucleolítica do exossomo de levedura, assim como o de archaea, é dependente da formação de heterodímeros; Ylr022p, uma proteína de levedura com função não caracterizada, liga inespecificamente RNA in vitro, mas mais eficientemente alguns RNAs in vivo. Dentre as proteínas de archaea, Pab418p e aNip7p também ligam RNA, e como demonstrado aqui, aNip7p influencia significativamente a atividade do exossomo de archaea. / The synthesis of ribosomes is one of the major metabolic pathways in eukaryotic cells. This process starts in the nucleolus and ends with the export and final maturation of the ribosomal subunits 40S and 60S in the cytoplasm. Three eukaryotic ribosomal RNAs (18S, 5.8S and 25S) are synthesized as a 35S primary transcript (35S pre-rRNA), which is then processed by a complex and ordered series of nucleotide modifications and endo- and exonucleolytic cleavage reactions. These processing reactions depend on 170 proteins, 80 small nucleolar RNAs and specific pre-rRNA sequences. The trans-acting factors, that take part in the processing can be grouped as RNA-helicases, endonucleases, snoRNPs (small nucleolar ribonucleoprotein complexes) and exonucleases, including the exosome. The yeast exosome is composed of 10 essential proteins that function in the processing of rRNAs, snRNAs, snoRNAs and in the degradation of aberrant mRNAs. Recently, the archaeal exosome structure was determined, but no information is yet available on the regulation of the exosome function or on the possible role of the cofactors that transiently interact with it. The main goals of this work were the functional characterization of the protein components of the Saccharomyces cerevisiae exosome RNase PH ring, as well as the structural and functional characterization of the possible cofactors of that complex, Nop17p and Ylr022p. Since the recent characterization of the Pyrococcus exosome, the study of the archaeal exosome cofactors, Pab418p, Pab1135p and aNip7p, was also included in this work, in order to correlate the data on the complex of these different organisms. Our results show that the exonucleolytic activity of the yeast exosome is dependent on the heterodimers formation, as described for archaea. Although it is not clear how Nip7p affects the exosome function in yeast, aNip7p binds RNA and inhibits a-exosome activity in vitro. Yeast Ylr022p binds RNA inespecificaly in vitro, but coprecipitates specific RNAs more efficiently from total cell extracts. Its archaeal orthologue, Pab418p, also binds RNA, but does not affect significantly a-exosome function.