Loss of the promyelocytic leukemia (PML) protein is associated with genomic instability/cancer. There are several isoforms of the PML protein that localize in PML nuclear bodies (PML NBs). How each individual isoform contributes to the functions of PML NBs is unknown. The objective of this study was to identify and characterize PML isoform-I (PML-I) specific protein-protein interactions. Using yeast two-hybrid screens, several interacting partners of PML-I were identified that play roles in translational regulation, including eukaryotic initiation factor 3 subunit K (eIF3K). Our studies demonstrated that eIF3K interacts with PML-I in vitro and in vivo. Through its interaction with eIF3K, overexpression of PML-I resulted in the concomitant increase in eIF3K protein levels in mammalian cells. This suggests that PML-I may be involved in regulating eIF3K protein translation or stability, which in turn could affect translation of specific mRNAs or global translation in cancer cells with reduced expression of PML-I.

Eukaryotic initiation factor-4E (eIF4E) controls the rate of cap-dependent translation initiation and is in turn exquisitely regulated by 4E-BPs. 4E-BP2 binds eIF4E with the highest affinity and is implicated in cancer, and metabolic and neurological disorders. Herein we use NMR, ITC and fluorescence to characterize 4E-BP2 structural and binding properties. Isolated 4E-BP2 is intrinsically disordered, but possesses some transient secondary structural propensities. eIF4E, however, is folded but has a disordered N-terminus. The eIF4E:4E-BP2 interaction is tight (Kd = 10-9 nM) and involves 4E-BP2 C-terminal and canonical binding regions, and the disordered eIF4E N-terminus. 4E-BP2 remains largely disordered upon binding to eIF4E. Noteworthy, high affinity interactions are not necessarily mediated by static structures, and 4E-BP2 binding is not the simple “disorder-to-order” transition observed in many interactions involving disordered proteins. This study offers molecular insights into 4E-BP2 functionality, and lays a foundation for development of novel therapies for cancer and neurological disorders.

Eukaryotic initiation factor-4E (eIF4E) controls the rate of cap-dependent translation initiation and is in turn exquisitely regulated by 4E-BPs. 4E-BP2 binds eIF4E with the highest affinity and is implicated in cancer, and metabolic and neurological disorders. Herein we use NMR, ITC and fluorescence to characterize 4E-BP2 structural and binding properties. Isolated 4E-BP2 is intrinsically disordered, but possesses some transient secondary structural propensities. eIF4E, however, is folded but has a disordered N-terminus. The eIF4E:4E-BP2 interaction is tight (Kd = 10-9 nM) and involves 4E-BP2 C-terminal and canonical binding regions, and the disordered eIF4E N-terminus. 4E-BP2 remains largely disordered upon binding to eIF4E. Noteworthy, high affinity interactions are not necessarily mediated by static structures, and 4E-BP2 binding is not the simple “disorder-to-order” transition observed in many interactions involving disordered proteins. This study offers molecular insights into 4E-BP2 functionality, and lays a foundation for development of novel therapies for cancer and neurological disorders.

Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2006. / Title from second page of PDF document. Includes bibliographical references (p. 160-176).

Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2004. / Title from second page of PDF document. Includes bibliographical references (p. 139-145).

The studies reported in this thesis describe the work done in the area of translation initiation where a previously unknown role of multiple copies of initiator tRNA in E. coli has been reported. Also the role of SsrA resume codon in resumption of translation, until not clearly known has been reported here. Chapter -1 discusses the relevant literature in understanding translation and initiator tRNA selection on the ribosome during initiation. It also discusses the literature pertaining to the aspect of release of stalled ribosomal complexes by SsrA. This is followed by the next chapter (chapter- 2) which discusses the materials and methods used throughout the study. Chapter- 3 describes the studies leading to the role of multiple copies of initiator tRNA in E. coli in governing the fidelity of initiator tRNA selection on the P site of the ribosome. This is followed by Chapter-4 which describes the role of the resume codon of the SsrA in governing the efficiency of trans-translation in releasing the stalled ribosomal complexes. The summaries of the chapters 3 and chapter 4 are briefly described below. i) Role of conserved 3GC base pairs of initiator tRNA in the initiator-elongator tRNA discrimination. Translation initiation is the first step in the very important and highly conserved biological process of protein biosynthesis. The process involves many steps, a wide array of protein factors at each specialized step and a large ribonucleoprotein particle; the ribosome to decode the information of the mRNA template into biologically active proteins. The process of initiation is still unclear largely due to fewer reports of available structural data. One of the very interesting questions that people have been trying to address is how the initiator tRNA is selected on the P- site of the ribosome and what is the importance of the conserved three GC base pairs in the anticodon stem of the initiator tRNA. Here in this study, I have studied this question by using the classical genetic technique of generating and characterizing the mutant initiator tRNA defective at the step of initiation. I have identified and analyzed the suppressors which are capable of rescuing this defect in initiation. The study involves two such E. coli suppressor strains (named D4 and D27). These suppressors can initiate translation from a reporter CAT mRNA with amber codon, independent of the presence of the three consecutive GC base pairs in the anticodon stem of initiator tRNAs. Mapping of the mutations revealed that the mutants are defective in expression of the tRNA1fMet (metZVW) gene locus which encodes the initiator tRNA. Both the suppressors (D4 and D27) also allow initiation with elongator tRNA species in E. coli. Taken together, the results show that E. coli when deficient in the initiator tRNA concentration can lead to initiation with elongator tRNA species. ii) The Role of SsrA/tmRNA in ribosome recycling and rescue. Occasionally during the process of translation, the ribosomes stall on the mRNA before the polypeptide synthesis is complete. This situation is detrimental to the organism because of the sequestration of the tRNAs as ‘peptidyl tRNAs’ and the ribosomes. In E. coli one of the pathways to rescue stalled ribosomes involves disassembly of these stalled complexes to release peptidyl tRNAs which are then recycled by peptidyl tRNA hydrolase (Pth), an essiential enzyme in E. coli. The other pathway which is not essential in E. coli but is conserved in all prokaryotes involves SsrA or tmRNA (transfer messenger RNA). The tmRNA is charged with alanine and recognizes the stalled ribosomal complexes and acts as tRNA to bind the A-site. It also functions as mRNA by adding a undecapeptide (which is actually a tag for degradation by cellular proteases) to the existing polypeptide and there is normal resumption of the translation. In most sequences of SsrA ORF, the first codon of the ORF, called as resume codon, is conserved. I wanted to understand the importance of the conservation of the resume codon. Towards this end I randomly mutated the resume codon and studied the effect of the altered resume codon in the rescue of stalled ribosomal complexes. The effect of over-expression of these mutants was investigated in the rescue of the Pthts defect since it is known that the overexpression of SsrA rescues the temperature sensitive phenotype of the Pthts strain and so causes less accumulation of peptidyl–tRNA in E. coli .The effect for these mutants has also been studied by the growth of hybrid λimmP22 phages. I also used AGA minigene system to study the effect of various mutants which has been shown to sequester tRNAArg (UCU) in the ribosomal P-site, translation of this minigene causes toxicity to E. coli. I have tried to study the effect of the SsrA mutants in rescue of toxicity caused by the minigene. Overall, the observations indicate that the conservation of the resume codon is important in E. coli and having mutated resume codon probably leads to deficient trans-translation during one or the other growth conditions.

Mass production of translationally optimized bacteriophages (hereafter referred to as phages) is the need of the hour in the application of phages to therapy. Understanding translational efficiency of phages is the major preliminary step for mass producing efficient phages. The objective of this thesis is to understand factors affecting translational efficiency of phages. In chapter two, we hypothesized that weak translation initiation efficiency is responsible for weak codon concordance of Escherichia coli lambdoid phages with that of their hosts. We measured the strength of translation initiation using two indices namely minimum folding energy (MFE) and proportion of Shine-Dalgarno sequence (PSD). Empirical results substantiate our hypothesis suggesting lack of strong selection for improving codon adaptation in these phages is due to their weak translation initiation. In chapter three, we measured codon usage concordance between GC-rich and GC-poor Aeromonas phages with their GC-rich host Aeromonas salmonicida. We found low codon usage concordance in the GC-poor Aeromonas phages. We were interested in testing for the role of tRNAs in the GC-poor phages. We observed that the GC-poor phages carry tRNAs for codons that are overused by the phages and underused by the host. These findings suggest that the GC-poor Aeromonas phages carry their own tRNAs for compensating for the compositional difference between their genomes and that of their host. Previously several studies have reported observed avoidance of stable secondary structures in start site of mRNA in a wide range of species. We probed the genomes of 422 phage species and measured their secondary structure stability using MFE. We observed strong patterns of secondary structure avoidance (less negative MFE values) in the translation initiation region (TIR) and translation termination region (TTR) of all analyzed phages. These findings imply selection is operating at these translationally important sites to control stable secondary structures in order to maintain efficient translation.

The conserved secondary structural RNA motifs of EncephaloMyoCarditis Virus (EMCV) have been well characterised biochemically and shown to play an important role in translation initiation by a novel cap-independent mechanism called Internal Ribosomal Entry Site (IRES). However, the three dimensional structure and interactions of these conserved motifs are not known, and hence the mechanism is not fully understood. The NMR results described in this thesis have provided, for the first time, new structural knowledge on the conformation of these motifs, their affinity for Mg2+ and their intermolecular interactions. RNA motifs selected from two separate domains (I and J) of the IRES structure were investigated using a range of 2D and 3D NMR techniques. The apical ‘hammerhead’ region of the I domain contains a highly conserved 16mer RNA which hosts a stable and mutationally sensitive G547CGA550 tetraloop. Sequence specific assignments were carried out on this motif, along with its Mg2+ complex, and a large number of NMR experimental constraints were generated for the RNA structure determination. Similarly, high resolution NMR structures of a distal 17mer RNA, which has been predicted to be a potential receptor for the GCGA tetraloop, and its Mg2+ complex were also produced. Thus, we were able to demonstrate that Mg2+ stabilises the RNA tertiary structure via non-specific interactions. Since the largest changes were induced at the tetraloop motif, we propose that Mg2+ stabilises the 16mer into an optimum conformation which is essential for IRES function. The determination of the structures of the above motifs led us to investigate the 16mer-17mer binary (1:1) complex at 1 GHz, in the presence of Mg2+. Significant changes were observed in the 1H and 31P chemical shift, NOE intensity and line width, clearly demonstrating RNA-RNA interactions taking place between the two components. The most interesting result to emerge was the distinct absence of NOEs from G547{NH} of the stable tetraloop, thus highlighting an important structural role for this functionally critical residue. Since no previous work has shown a clear interaction between the two RNAs, the results obtained in this project provide the first direct experimental evidence for intramolecular interactions in the I domain of EMCV IRES.Finally, we show how isotopically labelled RNAs can be successfully used as an aid in NMR assignment, analysis and structure determination. The J domain of EMCV IRES binds to eIF4GII protein and is essential for translation initiation. A suite of 3D NMR techniques were carried out on a highly enriched and uniformly 13C, 15N-labelled 39mer RNA. Several key features of the RNA, which may be involved in protein recognition, were identified. Further, a selectively 19F-labelled 16mer RNA from the I domain, was also studied to show how fluorine NMR can be used to probe RNA structure, dynamics and interactions. The RNA motifs of the EMCV IRES were shown to exhibit high stabilities, which are brought about by the complex folding of the various secondary structural elements involving RNA- Mg2+, RNA-RNA and RNA-protein tertiary interactions. It is these vital interactions that enable the IRES to recruit the ribosome in the translation initiation step of protein synthesis, and have laid a strong foundation for further NMR investigation of the whole IRES.

In plants, translation regulation plays an important role during progamic phase, fertilization and seed development. The process of translation is mostly regulated in its initiation phase, where Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex initiation factor, consisting of 12 different subunits. In plants, single eIF3 subunit mutants caused various growth and development defects, depending on the particular subunit that was mutated. However, not all the plant eIF3 subunits were characterized to this date. The objective of this work was to functionally characterize the eIF3 subunit A using Arabidopsis thaliana as the main model plant. We described in this work that plant eIF3A proteins share high levels of homology and domain organization with eIF3A subunits from non-plant eukaryotic species but contain regions specific only to plants. Next we described that Arabidopsis thaliana AteIF3A gene is transcribed in highly proliferating tissues, its protein product localizes to cytoplasm and around pollen vegetative cell nucleus and observed an increased frequency of defective pollen grains and defects in seed formation in plants with T-DNA insertion localized to the AteIF3A gene. We also produced stable transgenic Nicotiana tabacum lines expressing heterologous AteIF3A...

Machine-learning (ML) techniques have been widely applied to solve different problems in biology. However, biological data are large and complex, which often result in extremely intricate ML models. Frequently, these models may have a poor performance or may be computationally unfeasible. This study presents a set of novel computational methods and focuses on the application of genetic algorithms (GAs) for the simplification and optimization of ML models and their applications to biological problems. The dissertation addresses the following three challenges. The first is to develop a generalizable classification methodology able to systematically derive competitive models despite the complexity and nature of the data. Although several algorithms for the induction of classification models have been proposed, the algorithms are data dependent. Consequently, we developed OmniGA, a novel and generalizable framework that uses different classification models in a treeXlike decision structure, along with a parallel GA for the optimization of the OmniGA structure. Results show that OmniGA consistently outperformed existing commonly used classification models. The second challenge is the prediction of translation initiation sites (TIS) in plants genomic DNA. We performed a statistical analysis of the genomic DNA and proposed a new set of discriminant features for this problem. We developed a wrapper method based on GAs for selecting an optimal feature subset, which, in conjunction with a classification model, produced the most accurate framework for the recognition of TIS in plants. Finally, results demonstrate that despite the evolutionary distance between different plants, our approach successfully identified conserved genomic elements that may serve as the starting point for the development of a generic model for prediction of TIS in eukaryotic organisms. Finally, the third challenge is the accurate prediction of polyadenylation signals in human genomic DNA. To achieve this, we analyzed genomic DNA sequences for the 12 most frequent polyadenylation signal variants and proposed a new set of features that may contribute to the understanding of the polyadenylation process. We derived Omni-PolyA, a model, and tool based on OmniGA for the prediction of the polyadenylation signals. Results show that Omni-PolyA significantly reduced the average classification error rate compared to the state-of-the-art results.