Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: Mary C. Farach-Carson, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Plant Biology." Includes bibliographical references.

Thesis (Ph.D.) -- University of Adelaide, Department of Genetics, 1977.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.

Translational efficiency is dictated in part by the availability of charged transfer RNA. Depletion of aminoacylated tRNAs (e.g. during recombinant protein expression) can increase translational errors and associated stress responses. Here, the role of tRNAs as regulators of gene expression was explored through development of synthetic, tRNA-regulated gene circuits, and through an investigation of the impact of tRNA aminoacylation on endogenous gene expression. Synthetic gene circuits initially explored the use of dominant negative alleles of the release factor eRF1 to modulate stop codon readthrough and translationally regulate gene expression. Mutant eRF1 proteins exhibited only a six-fold stimulatory effect on stop codon readthrough. The dominant negative phenotype was rescued partially by overexpression of eRF1, but not eRF3. Ultimately the severity of growth inhibition by these eRF1 alleles limited their utility in synthetic gene circuit design. A novel synthetic circuit was then implemented that utilised TetR interaction with a TetR-inducing peptide in order to control the expression of a suppressor tRNA, and thus a luciferase reporter gene. Using a parameterised mathematical model, the promoter configuration of the circuit was successfully optimised, allowing suppressor tRNAs to regulate the production of luciferase in both feedforward and positive feedback modes of operation. The effects of charged tRNA levels on the global translation network were dissected by regulating the S.cerevisiae glutamine tRNA synthetase gene GLN4 using a tet-off doxycyclineregulated promoter. tRNA synthetase depletion caused the activation of the Gcn4 amino acid starvation response due to accumulation of uncharged glutamine tRNAs. Doxycycline GLN4 shut-off caused increased amino acid production, and decreased ribosome biosynthesis at the transcriptomic and proteomic level, and further physiological changes proposed to result from compromised translation of glutamine-rich regulatory proteins. tRNA overexpression in the GLN4 depletion strain successfully caused altered competition between different isoacceptor tRNA types for their cognate synthetase resource. Together, these results support a growing understanding of tRNA as a key modulator of translation and gene expression in synthetic and natural systems.

No description available.

Protein synthesis is an essential process for all living organisms and is an effective major target for current antibiotics. Elongation factor Tu (EF-Tu) is a highly conserved and essential protein that functions during protein synthesis. EF-Ts interacts with EF-Tu to help maintain a functionally active state of EF-Tu required for cell growth. Although EF-Ts is essential for Escherichia coli, its sequence is poorly conserved. LepA is a highly conserved protein within bacteria and has a similar structure to EF-Tu. In spite of this, LepA has been shown to be non-essential under ideal conditions and the function of LepA still remains elusive. An analysis on the structurally unique aspects of LepA, EF-Tu and EF-Ts was performed here in an effort to gain an understanding on the functions of these proteins. This knowledge, in combination with their unique structural components will provide important tools in developing new and effective antibiotics. / xiii, 177 leaves : col. ill. ; 29 cm

We have studies oxidative damage of RNA, a major type of cellular macromolecules. RNA is a primary target of reactive oxygen species (ROS). Under oxidative stress, most nucleic acid damages in Escherichia coli (E.coli) are present in RNA as shown by high levels of 8-oxo-G, an oxidized form of guanine. Increased RNA oxidation is closely correlated to cell death under oxidative stress. Surprisingly, neither RNA structure nor association with proteins protects RNA from oxidation... Our results demonstrate a major role for RNA degradation in controlling oxidized RNA. We have identified activities that may work in specific pathways for selectively degrading damaged RNA. These activities may play pivotal rold in controlling oxidized RNA and protecting cells under oxidative stress. / by Min Liu. / Thesis (Ph.D.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Translation is the process by which the cell produces new proteins on the ribosome, as directed by genetic instructions, in all living organisms. Structural studies of the ribosome have shed considerable lights on its mechanism and regulation. Cryogenic electron microscopy (cryo-EM) and single-particle reconstruction technique is one of the major approaches to studying ribosome structure. In this thesis, I report the use of cryo-EM and related new techniques to study the structure of ribosome complexes. This work is divided into three parts. First, in Chapter 3, I describe the development of a computational method in the classification of cryo-EM data. Recently developed classification methods have enabled resolving multiple structures/conformations of the molecules from cryo-EM data obtained on a heterogeneous biological sample. However, the classification methods all involve various amounts of arbitrary decisions made by researchers, which can limit the use of these methods by inexperienced users. As a step toward fully automated classification, I worked with colleagues to develop a "jumper analysis" to determine the number of distinguishable classes of 3D reconstruction, based on the statistics of cryo-EM particles. Second, in Chapter 4, I document the cryo-EM study of EttA-70S ribosome complex, which provided structural insights into the mechanism of EttA in translation regulation. Energy-dependent translation throttle A (EttA, previously named YjjK in Escherichia coli) is the most prevalent member of ATP-binding cassette F family proteins in eubacteria. Through a collaboration among the Hunt, Frank, and Gonzalez labs, we combined crystallography, biochemical, cryo-EM and single-molecule fluorescence energy transfer techniques to elucidate the function and mechanism of EttA. We demonstrated that EttA gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. We also showed that the ATP-bound form of EttA binds to the ribosomal tRNA-exit site, and restricts the ribosome and tRNA dynamics required for translation. Thirdly, in Chapter 5, I discuss the improvements to a new technique, time-resolved cryo-EM by mixing-spraying, and its application to ribosome studies. The mixing-spraying method can study processes involving two big biological molecules that are in the sub-second time scale. I worked with colleagues to apply this method to studying ribosome subunit association. By mixing the subunits and reacting for 60 ms and 140 ms, we were able to capture the association reaction in a pre-equilibrium state. We detected three 70S ribosome conformations in the system. Quantification of the proportions of particles assuming these conformations suggested that the 70S ribosome can undergo fast conformational changes upon formation, and reaches equilibrium among these conformations earlier than 60 ms. In addition, I present preliminary results of studying translation decoding using the mixing-spraying method. This study, performed before improving the mixing-spraying method, was inconclusive mainly due to the limited size of cryo-EM data. Now that we have demonstrated the capability of the mixing-spraying method to visualize multiple states of molecules in a sub-second reaction, the translation decoding process can be revisited and many other processes, such as translation initiation, can be studied.

Translation of an mRNA template into its corresponding protein is necessarily a highly accurate process. In all organisms, this translation is performed by the universally conserved macromolecular machine, the ribosome. However, the mechanisms through which the ribosome is able to regulate translation, and therefore ensure its fidelity, are not well understood. Often these types of mechanisms, which ensure molecular fidelity, utilize multiple, transient states over which cognate and non-cognate substrates are discriminated multiple times. However, such transient and/or rarely populated states are difficult to study by conventional, ensemble experimental techniques. In this thesis, single-molecule fluorescence resonance energy transfer (smFRET), which alleviates many of these limitations, is used in order to interrogate the dynamics of a translation factor, release factor 1 (RF1), and how they are organized to ensure accurate and efficient recognition of stop-codons during the termination stage of translation. In order to observe the dynamics of the RF1 binding and codon discrimination processes with smFRET, a relatively high concentration of fluorophore-labeled RF1 must be used in order to observe significant binding to sense-codons; however, such high concentrations are not accessible with traditional smFRET total internal fluorescence microscopy. Therefore, in Chapter 2 a novel approach to breaking this concentration barrier is presented, in which robustly-passivated gold-based nanoaperture arrays are developed to limit the excitation volume used in smFRET measurements of RF1. Unfortunately, as in the case of RF1 binding to sense-codon programmed ribosomes, many of the ribosomal dynamics that are in principal observable using smFRET are too fast to observe using current wide-field detectors. Therefore, Chapter 3 investigates the precision and accuracy with which transient conformational dynamics can be quantified using single-molecule techniques such as smFRET. As a case study, these approaches were used to analyze the dynamics of the GS1-GS2 equilibrium of the pretranslocation (PRE) ribosome--a situation where transient intermediate states that can be observed using single-particle cryo-electron microscopy are not seen using smFRET. In Chapter 4, a novel computational method is developed to address such temporally-limited single-molecule data, and in doing so, it is used to analyze the structural contributions of tRNA to ribosomal transition state energy barriers using temperature-dependent smFRET with temporal super-resolution. The temperature-dependence of reaction rate constants is governed by the underlying thermodynamic landscape of the molecular system. To investigate the energy landscape over which the PRE ribosome operates, temperature-dependent smFRET experiments were performed on PRE complexes containing different tRNAs. By investigating the relative temperature-dependence of the rate constants involved in the GS1 - GS2 equilibrium as a function of tRNA identity, nascent polypeptide chain presence, and A and P site occupation, relative thermodynamic contributions of the different structural elements were quantified. Unfortunately, this investigation was complicated by fast rate constants which approach the time resolution limitations of smFRET TIRF experiments, especially with the increased temperatures used in these experiments. Additionally, it is complicated by the heterogeneity within the ensemble of ribosomes that is created when some of the enzymatically-prepared ribosomal complexes fail to undergo, or undergo additional rounds of translation. To overcome these complications, a novel computational method to achieve temporal super-resolution. This method uses Bayesian inference for the analysis of sub-temporal resolution data (BIASD). By integrating this approach with a Bayesian variational mixture model, the fast dynamics of heterogenous populations can be accurately and precisely quantified. This then allowed the contributions of the structural differences that the various tRNA make to the underlying PRE complex energy landscape to be determined. The conformational dynamics that regulate the binding affinity and codon discrimination ability of RF1 are investigated in Chapter 5. During the elongation stage of translation, class I release factors compete with aminoacyl-tRNAs to interrogate the mRNA triplet-nucleotide codon that is located in the ribosomal aminoacyl-tRNA binding (A) site. To avoid deleterious effects, class I RFs must be able to accurately discriminate stop-codons from sense-codons, only triggering the termination stage of translation and catalyzing the release of the nascent polypeptide chain from the peptidyl-tRNA located in the ribosomal peptidyl-tRNA binding (P) site upon recognition of a stop-codon. Despite its importance for ensuring the accuracy of gene expression, the high fidelity mechanism through which class I RFs discriminate sense codons remains elusive. Using smFRET, the kinetics with which a fluorophore-labeled, bacterial RF1 binds to the A site of bacterial ribosomal release complexes carrying a fluorophore-labeled peptidyl-tRNA in the P site and either a stop-codon, or a sense-codon that differs from a stop-codon by a single nucleotide (i.e., a near-stop codon) programmed in the A-site are investigated. The results of these experiments, as well as analogous experiments performed using RF1 mutants or antibiotic inhibitors of RF1 function, reveal that RF1 binding affinity and codon discrimination occurs via a multistep process. Taken together with molecular dynamics simulations of wildtype and mutant RF1, these data demonstrate how the conformation dynamics of the switch loop modulate RF1 binding affinity and codon discrimination--enabling the elucidation of some of the molecular details through which class I RFs ensure the integrity of translation elongation and the fidelity of translation termination.