Using the Gillespie algorithm, the export of the mRNA molecules from their transcription site to the nuclear pore complex is simulated. The effect of various structures in the nu- cleus on the efficiency of export is discussed. The results show that having some of the space filled by chromatin near the mRNA synthesis site shortens the transport time. Next, the complete eukaryotic gene expression including transcription, splicing, mRNA export, translation, and mRNA degradation is modeled using delay stochastic simulation. This allows for the study of stochastic effects during the process and on the protein production rate patterns. Various protein production patterns can be produced by adjusting the poly-A tail length of the mRNA and the promoter efficiency of the gene. After that, the opposing effects of the chromatin density on the seeking time of the transcription factors for the promoter and the exit time of the mRNA product are discussed. / xi, 102 leaves ; 28 cm

DNA is the genetic material of a cell and is copied in the form of pre-mRNA through transcription in eukaryotes. RNA polymerase II is responsible for the transcription of all genes that express proteins. Transcription is a significant source of the stochasticity in gene expression. In this thesis, I discuss the development of a biochemically detailed model of eukaryotic transcription, which includes pre-initiation complex (PIC) assembly, abortive initiation, promoter-proximal pausing and termination as the points that can be slow steps for transcription. The stochastic properties of this model are studied in detail by stochastic simulations with some preliminary mathematical analysis. The results of this model suggest that PIC assembly can play the most significant role in affecting the transcription dynamics. In addition, promoter-proximal pausing has been identified as a potential noise regulatory step in eukaryotic transcription. These results show excellent agreement with many experimental studies. / x, 107 leaves : ill. ; 29 cm

Translation initiation factor 5A (IF5A) is an essential, highly conserved protein found within all eukaryotic (eIF5A) and archaeal (aIF5A) cells. The IF5A protein is unique in that it contains the amino acid hypusine; a two-step post translational modification of a single, conserved lysine residue. Although hypusination of eIF5A is vital for eukaryotic cell viability, the primary role of the protein and its hypusine side chain remain a mystery. eIF5A, initially identified as a translation initiation factor, is not required for global protein synthesis leading to the prevailing proposal that eIF5A is purely involved in the translation of a select subset of mRNAs. Recently a number of mutational studies have focused on the conserved, hypusine-containing loop region of eIF5A where specific residues have been found to be essential for activity without affecting hypusination. It has been postulated that eIF5A exists as a dimer (40 kDa) under native conditions and that these residues may be at the interface of dimerisation. The aim of this research was therefore to conduct a mutational analysis of the loop region in support of this hypothesis. A functional analysis of the Saccharomyces cerevisiae eIF5A mutant proteins K48D, G50A, H52A and K56A revealed that these substitutions impaired growth to varying degrees in vivo with G50A and K48D mutant proteins displaying the most convincing defects. Gel filtration profiles gave unexpected results determining eIF5A mutant and wild type proteins to have a native molecular weight of 30 to 31 kDa, suggesting that the eIF5A oligomeric state may be transitory and subject to certain conditions. The inconclusive results obtained from using gel filtration studies led to an investigation into the feasibility of producing native, hypusinated peptides for future structural studies using nuclear magnetic resonance. Hypusinated and unhypusinated eIF5A were successfully separated into their domains making this a possibility. Finally, this study proposes a role for eIF5A in eukaryotic IRES-driven translation initiation.

Indiana University-Purdue University Indianapolis (IUPUI) / Translation Initiation in protein synthesis is a crucial step controlling gene expression that enhanced by eukaryotic translation initiation factors (eIFs). eIF3a, the largest subunit of eIF3 complexes, has been shown to regulate protein synthesis and cellular response to cisplatin treatment. Its expression has also been shown to negatively associate with prognosis. In this study, we tested a hypothesis that eIF3a regulates synthesis of proteins important for repair of double strand DNA breaks induced by ionizing radiation (IR). We found that eIF3a up-regulation sensitizes cellular response to IR while its knockdown causes resistance to IR. We also found that eIF3a over-expression increases IR-induced DNA damage and decreases Non-Homologous End Joining (NHEJ) activity by suppressing expression level of NHEJ repair proteins such as DNA-PKcs and vice versa. Together, we conclude that eIF3a plays an important role in cellular response to DNA-damaging treatments by regulating synthesis of DNA repair proteins and, thus, eIIF3a likely plays an important role in the outcome of cancer patients treated with DNA-damaging strategies including ionizing radiation.