Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of California, San Francisco, 2004. / Includes bibliographical references. Also available online.

Viruses require a host cell to replicate and proliferate; upon infection they appropriate host resources and molecular machines. Specifically, viruses use ribosomes of the host to translate the information in their genome. Some viruses with single-stranded RNA genomes contain highly structured non-coding regions of RNA called internal ribosome entry sites (IRESs) which are used to hijack the host’s ribosomes through a non-canonical cap-independent initiation pathway. Canonical translation initiation is a highly complex and regulated process: at least a dozen translation factors are necessary, and it is the rate-limiting step in eukaryotic translation. Viruses containing an IRES forgo canonical eukaryotic translation initiation factors and bypass some steps of canonical translation initiation by mimicking part of the host’s initiation machinery. The simplest among these IRESs are found in the intergenic region (IGR) of viruses in the family Dicistroviridae. These type IV IRESs from dicistroviruses have been structurally characterized in great detail in using the cricket paralysis virus (CrPV) and Israeli Acute Paralysis Virus (IAPV). To better understand how structure affects the function of these type IV IRESs, using single-particle cryo-electron microscopy (cryo-EM), we have characterized a recently discovered IRES found in the IGR of the genome of Nedicistrovirus (NediV). Four complexes that represent each step in the alternative translation initiation mechanism were prepared and analyzed to solve the 3D structure and characterize the mechanism by which the NediV-IRES captures host ribosomes. With this, we were able to understand how the shorter stem-loop V (SL-V) of NediV-IRES impacts the well-characterized interaction of SL-V with eukaryotic small subunit ribosomal protein 25 (eS25) (Landry et al., 2009), which is important for the IRES:40S complex formation. This shortened stem-loop has been shown to fold in a way that does not support stable binding to the small ribosomal subunit (40S) and subsequent recruitment of the large ribosomal subunit (60S). NediV-IRES, rather, relies on direct recruitment of the 80S ribosome, which has been seen more commonly at low concentrations of Mg²⁺ for CrPV-IRES (Petrov et al., 2016). Solved structures also suggest that upon loading, NediV-IRES skips the first eEF2-dependent pseudo-translocation step necessary to bind to the ribosomal P site without the need of eEF2. Because of their simplicity, these type IV IRESs represent a robust potential tool for cell-free and vector-driven translation. Due to these structural and mechanistic differences observed, we propose that NediV-IRES, along with the NediV-like Antarctic picorna-like virus 1 (APLV-1)-IRES (Lu, 2019), represents a novel type IV IRES subclass.

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Protein kinase CK2 phosphorylates wheat eIF2, eIF3, eIF4B, eIF5 and three 60S ribosomal proteins. The substrate specificity of CK2[alpha] toward various plant initiation factor substrates was altered in vitro through holoenzyme formation in the presence of regulatory [beta]-subunits. This presents a potential mechanism through which the differential expression and sub-cellular distribution of CK2 [beta]-subunits could regulate phosphorylation of various CK2 substrates in plants. Our analysis of initiation factor phosphopeptides produced by in vitro phosphorylation identified 20 CK2 phosphorylation sites in eIF2[alpha], eIF2[beta], eIF3c, eIF4B, and eIF5. Native wheat eIF5 was prepared in the presence of phosphatase inhibitors and analyzed by mass spectrometry. Native wheat eIF5 was determined to be a phosphoprotein containing at least 3 phosphorylation sites. The C-terminal CK2 site (S451) of native eIF5 was completely phosphorylated, and tryptic fragments containing the other in vitro CK2 two sites (S209, T240) also appear to be partially phosphorylated. Many of the CK2 phosphorylation sites identified are in conserved binding domains of the yeast multifactor complex (eIF1/eIF3/eIF5/eIF2/GTP/Met-tRNAi[superscript Met). This observation lead to the hypothesis that CK2 phosphorylation may regulate the formation of plant multifactor complexes. The results presented here suggest that plant initiation factors are capable of forming complexes similar to those previously reported in yeast. The in vitro interaction of initiation factors within these complexes appears to be enhanced by phosphorylation of eIF2, eIF3c, and eIF5 by CK2. Site-directed mutagenesis of eIF5 to remove CK2 phosphorylation sites not only prevents the CK2 mediated increase in interaction with eIF1, but also resulted in reduced stimulation of translation initiation in vitro. / text

During protein synthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of mRNA-programmed ribosomes in a GTP-dependent manner. To enable future studies on the functional and structural requirement of EF-Tu’s function, a Cysteine-free variant of EF-Tu was constructed suitable for subsequent labelling of the protein and use in kinetic studies. Here, the kinetic properties of three Cysteine-less EF-Tu variants are reported, demonstrating that only the variant with the Alanine substitution in position 81 retains wild-type activity with respect to the interaction with guanine nucleotides, aa-tRNA and the ribosome. To explore a possible tRNA independent pathway for the GTPase activation signal, three residues in domain II of EF-Tu (Arginine 279, Glycine 280, Arginine 283) were mutated; the activity of EF-Tu variants were analyzed. Results suggest that these residues are indeed required for efficient ribosome-dependent stimulation of the GTPase activity of EF-Tu. / x, 85 leaves : ill. (some col.) ; 29 cm

The Drosophila melanogaster eIF4E binding protein (d4E-BP) inhibits translation initiation and is implicated in cell growth as a downstream effector of the Drosophila insulin signaling pathway. Since d4E-BP null flies show similar growth and development to control flies, the possibility of a conditional phenotype was explored through stress treatments. Adult d4E-BP null flies show sensitivity to oxidative stress, and d4E-BP null larvae die faster than controls under starvation and protein starvation. Expressing a mutant d4E-BP that doesn't bind to eIF4E in the d4E-BP null background does not rescue this stress sensitivity, which suggests that wild-type stress resistance requires binding of d4E-BP to eIF4E. / The Drosophila forkhead transcription factor dFOXO is a transcriptional activator of d4E-BP. There is a strong reduction of d4E-BP peptide in a dFOXO null background. dFOXO null flies are also sensitive to oxidative stress, and rescue of this sensitivity through ectopic expression of UAS-d4E-BP(wt) in a dFOXO null background suggests d4E-BP is a downstream mediator of dFOXO oxidative stress resistance.

Bibliography: leaves 167-176. / xvi, 176 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this thesis is to characterise a bacterial expression system for recombinant production of proteins with relevance to industry. Recombinant protein expression under control of stationary-phase inducible promoters is characterised, and the expression levels are quantitatively compared with those under control of IPTG-inducible promoters. A number of bacterial expression systems are constructed using promoter::cat transcriptional fusions. / Thesis (Ph.D.)--University of Adelaide, Depts. of Microbiology and Immunology and Chemical Engineering, 1997