The S1 genome segment of avian reovirus is functionally tricistronic, encoding three independent protein products (named p10, p17 and ?C) from three sequential, partially overlapping open reading frames (ORFs). The dogma of translation initiation, the cap-dependent scanning model, suggests that ribosomes would normally only translate the 5?-proximal ORF. Four alternate mechanisms of translation initiation could account for translation of the downstream ?C ORF; an IRES element, reinitiation, ribosome shunting, and leaky scanning. The objective of my doctoral research was to investigate the translation initiation mechanisms that are operative on the S1 mRNA. Translation of the p10 and p17 ORFs was revealed to be coordinated via standard leaky scanning, while none of the known mechanisms of translation initiation could account for expression of the ?C ORF. Further investigation determined that two alternate cap-dependent mechanisms contribute to translation initiation at the ?C AUG codon. The first mechanism involves a modified version of enhanced leaky scanning. Although insertion of upstream elements known to impede scanning ribosomal subunits dramatically inhibited translation of the downstream ORF in the context of other mRNAs, the same elements only marginally reduced ?C translation. Specific features of the S1 mRNA therefore function to promote leaky scanning and translation of the ?C ORF. The inability to eliminate ?C expression beyond a threshold retention level of ~20-30%, despite the presence of eight upstream start codons that should eliminate leaky scanning, strongly suggests that ribosomes must also utilize a scanning-independent means to access the internal ?C start site. This mechanism for ?C translation initiation, which I termed ribosome handoff, allows ribosomes to bypass upstream elements, and requires a sequence-dependent translation enhancer element present within S1 nucleotides 366-392 that may function to mediate handoff via complementarity with 18S ribosomal RNA. Translation initiation at the ?C start site is therefore made possible by two alternative mechanisms, enhanced leaky scanning and ribosome handoff from the 5?-cap. The novelty of these two mechanisms highlights the complexity of the translation initiation process and the potential heterogeneity of cellular ribosomes, which raises the possibility that internal initiation may be far more common than currently appreciated.

abstract: This work advances structural and biophysical studies of three proteins important in disease. First protein of interest is the Francisella tularensis outer membrane protein A (FopA), which is a virulence determinant of tularemia. This work describes recombinant expression in Escherichia coli and successful purification of membrane translocated FopA. The purified protein was dimeric as shown by native polyacrylamide gel electrophoresis and small angle X-ray scattering (SAXS) analysis, with an abundance of β-strands based on circular dichroism spectroscopy. SAXS data supports the presence of a pore. Furthermore, protein crystals of membrane translocated FopA were obtained with preliminary X-ray diffraction data. The identified crystallization condition provides the means towards FopA structure determination; a valuable tool for structure-based design of anti-tularemia therapeutics. Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination. Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2020