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Protein synthesis in all organisms is catalyzed by a highly-conserved ribonucleoprotein macromolecular machine known as the ribosome. Prior to each round of protein synthesis in the cell, a functional ribosomal complex is assembled from its component parts at the start site of a messenger RNA (mRNA) template during the process of translation initiation. In bacteria, rapid and high-fidelity translation initiation is promoted by three canonical initiation factors: IF1, IF2, and IF3. In this thesis, I report the use of single-molecule fluorescence methods to study the role of the initiation factors and ribosome-factor interactions in regulating molecular events that occur during late stages of the translation initiation pathway. In Chapter 1, I provide a structural and biochemical framework for understanding one of the key events of the initiation pathway: docking of the large (50S) ribosomal subunit with the small subunit 30S initiation complex (30S IC). The 50S subunit joining reaction is catalyzed by GTP-bound IF2 and results in formation of a 70S initiation complex (70S IC) that contains an initiator transfer RNA (tRNA) and is primed for formation of the first peptide bond. During 50S subunit joining, IF2-GTP establishes interactions with RNA and protein components of the 50S subunit's GTPase-associated center (GAC), which play an important role in subunit recruitment as well as the subsequent activation of GTP hydrolysis by IF2. In Chapter 2, I describe the development of a single-molecule fluorescence resonance energy transfer (smFRET) signal to monitor the interactions between IF2 and the ribosome's GAC during real-time 50S subunit joining reactions. Specifically, the role of the L11 region, comprising ribosomal protein L11 and its associated ribosomal RNA (rRNA) helices, was investigated. The L11 region is a prominent structural component of the GAC that is believed to undergo large-scale conformational changes during protein synthesis; however, the nature and timescale of these conformational dynamics, and their role in regulating the biochemical activities of IF2 during initiation, are not known. I demonstrate that my smFRET-based 50S subunit joining assay is sensitive to conformational rearrangements between IF2 and L11 within the 70S IC and can thus be used as a tool for characterizing GAC dynamics and elucidating their function during initiation. Furthermore, my smFRET approach is shown to provide information on the rate of 50S subunit joining as well as the rate of IF2 dissociation from the 70S IC. Notably, IF2-dependent GTP hydrolysis was found to influence the extent of 70S IC conformational dynamics as well as the dissociation rate of IF2. The role of IF3 in regulating 50S-subunit joining dynamics is discussed in Chapter 3. IF3 plays an important role in ensuring the fidelity of translation initiation by preventing the formation of initiation complexes containing a non-initiator tRNA and/or a non-canonical mRNA start codon. Inclusion of IF3 within the 30S IC in the smFRET experiments was found to render the IF2-catalyzed 50S subunit joining reaction highly reversible. Direct observation of repetitive docking and undocking of the 50S subunit with the 30S IC indicates that IF3 may modulate translation initiation efficiency by influencing the stability of the 70S IC. The individual 50S subunit docking events were found to result in the formation of very different classes of 70S IC, characterized by different stabilities and unique patterns of IF2-L11 interactions. I propose that these dynamics reflect an underlying conformational equilibrium of the IF3-bound 30S IC that is read out during 50S subunit joining, and that this equilibrium could be modulated in order to regulate the efficiency of translation initiation. Following initiation-factor mediated assembly of the 70S IC, the first aminoacyl-tRNA is delivered to the ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP. Accommodation of aminoacyl-tRNA into the ribosome's peptidyl transferase center leads to formation of the first peptide bond, which signals the end of initiation and entry into the elongation phase of protein synthesis. The ternary complex binding site on the ribosome overlaps with that of IF2 at the GAC; a question of key mechanistic importance in understanding how the ribosome coordinates the transition from initiation to elongation thus concerns the relative timing of ternary complex binding with respect to IF2 dissociation from the 70S IC. In Chapter 4, I present preliminary results from two- and three-color fluorescence co-localization experiments aimed at characterizing the timing of these events at the single-molecule level. The data strongly suggest the occurrence of simultaneous occupancy of the ribosome by IF2 and ternary complex, implying that the ribosome is structurally capable of recruiting ternary complex prior to IF2 release from the 70S IC. The observation that the ribosome can accommodate more than one translation factor at a time may have important implications for understanding how it efficiently coordinates factor binding and release throughout protein synthesis, and opens the door to mechanistic studies of the ribosomal L7/L12 stalk, presumed to play a prominent role in these processes.

Translational control is an important means of regulating gene expression. Development of the Drosophila germ line relies on translational regulation to differentially express maternal mRNAs, allowing it to develop distinctly from the soma. One of the critical factors required for germ cell development and function is the conserved DEAD-box RNA helicase Vasa (Vas). The research presented in this thesis examines the role of Vas in translational regulation during Drosophila germ line development. A two-hybrid screen conducted with Vas identified a translation initiation factor eIF5B (dIF2), as a direct interactor. Mutations were created in eIF5B and were found to enhance the vas mutant phenotypes of reduced germ cell numbers, and posterior segmentation defects, suggesting a functional interaction between these factors in vivo. In order to further understand the biological significance of the Vas-eIF5B interaction, the region of Vas required for eIF5B-binding was mapped and then specifically disrupted. Reduction of Vas-eIF5B binding using a transgenic approach, virtually eliminated germ cell formation, while having only a moderate effect on the somatic requirement of Vas in posterior segmentation. In addition, Vas-eIF5B interaction was found to be required for the establishment of polarity within the egg during oogenesis, likely through direct regulation of gurken (grk) mRNA. We concluded that through interaction with eIF5B, Vas plays a critical role in translational regulation in the germ line. In addition, another Drosophila DEAD-box protein, highly similar to Vas, called Belle (Bel) was characterized. Mutations in bel were found to also affect the germ line, leading to both female and male sterility. Like Vas, Bel is implicated in translation initiation, however bel is an essential gene, with a requirement for growth, whose function is not restricted to the germ line. Our data suggest that Bel may be a nucleocytoplasmic shuttling protein,

Thesis (M.S.)--University of Texas at El Paso, 2009. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Thesis (M.S.)--University of Alabama at Birmingham, 2007. / Description based on contents viewed Oct. 5, 2007; title from title screen. Includes bibliographical references (p. 38-42).

No description available.

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

Protein synthesis, one of nature's most fundamental processes within all living cells, is catalyzed by the ribosome, a highly conserved, massive, two-subunit ribonucleoprotein complex. Ribosomes synthesize proteins based on the sequence of triplet-nucleotide codons presented by the messenger RNA (mRNA) template, using aminoacyl-transfer RNAs (aa-tRNAs) substrates, which deliver individual amino acids to the ribosome. Recent biochemical, structural, dynamic and computational studies have uncovered large-scale conformational changes of the ribosome, its tRNA substrates, and translation factors that play important roles in regulating protein synthesis, especially during the elongation phase of translation. For example, translocation of the ribosome along its mRNA template involves several conformational rearrangements of the ribosomal pre-translocation (PRE) complex, including the rotation of two ribosomal subunits, closure of the L1 stalk element, and reconfigurations of the ribosome-bound tRNAs. Importantly, modulation of these conformational changes of PRE complexes is used as a strategy by the cell and ribosome-targeting antibiotics to regulate translation elongation. Therefore, a complete understanding of the conformational dynamics of ribosomal complexes will not only improve our knowledge on how translation is regulated, but also provide crucial information for designing next-generation antibiotics. This thesis presents efforts demonstrating several strategies the cell develops in order to regulate translation by modulating the conformational dynamics of ribosomal complexes. In Chapter 2, I investigate if and how the individual dynamics of intersubunit motion, tRNA and L1 stalk are coordinated within PRE complexes, so that the translocation reaction is facilitated. To address this question, the dynamics of ribosomal intersubunit rotation were predictably perturbed using either structurally guided ribosome mutagenesis as well as an ribosome-targeting antibiotic translation inhibitor. Correspondingly, I used two single-molecule fluorescence resonance energy transfer (smFRET) signals to directly monitor how perturbation of the dynamics of intersubunit rotation alter the dynamics of P-site tRNA and the L1 stalk in PRE complexes. Taken together with the results of my complementary in vitro biochemical assays, my smFRET work clearly demonstrates that the ribosome coordinates individual conformational changes to maximize and regulate the efficiency of the translocation reaction. It is very likely that this strategy is used by the ribosome in other steps during translation for efficient chemical or mechanical reactions, and is taken advantage of by translation factors and antibiotics as part of the mechanisms through which they regulate and inhibit translation, respectively. Energy-dependent translational throttle A (EttA) is one regulatory translation factor that has been recently discovered and characterized through a collaboration between the Hunt, Gonzalez, and Frank laboratories (Chapter 3). Biochemical experiments have shown that in the presence of a high ADP/ATP ratio, EttA inhibits formation of the first peptide bond, and such inhibition is relieved upon addition of ATP, indicating that EttA may regulate the synthesis of proteins in response to the energetic status of the cell, as reflected by the cellular ADP/ATP ratio. Complementary cryo-EM studies have shown that the ATP-bound form of EttA binds to the ribosome at the E-site from where it directly contacts and forms bridging interaction between the L1 stalk and P-site tRNA. The results of my smFRET experiments demonstrate that EttA differentially modulates the conformation and/or dynamics the L1 stalk, depending on whether EttA is bound to ADP or ATP, thereby providing a possible rationale for the distinct effects of EttA on dipeptide synthesis in the presence of ADP vs. ATP. My smFRET data, together with the biochemical and structural efforts, demonstrate that EttA functions, at least in part, by restricting ribosome and tRNA dynamics that are crucial for translation. More importantly, our data support a model for the interaction of EttA with the ribosomal complex and its regulation of translation at the start of the elongation cycle, the molecular mechanism of which EttA uses has never been found among all other known translational regulatory factors. +1 non-programmed ribosomal frameshifting (+1 FS), in which the elongating ribosome slips by one nucleotide towards the 3' end of the mRNA during translation, occurs at a low frequency as a translational error. Proline-tRNA with an anticodon GGG (tRNAProGGG) is prone to induce +1 FS, and the evolution of post-transcriptional modifications of tRNAProGGG is used as a strategy by nature to suppress this error. However, the mechanism underlying this suppression has not been previously characterized. tRNAProGGG modifications have been shown to play important roles in regulating the conformational stability and flexibility of the secondary and tertiary structures of tRNAs and therefore have the potential to regulate the conformational dynamics of ribosomal complexes during translation elongation. However, the effect of these modifications on elongating ribosomal complex dynamics is completely unknown, thus greatly impeding our understanding of the role that they play in maintaining the translational reading frame. Chapter 4 presents the efforts to elucidate the mechanism by which tRNA modifications suppress +1 FS by investigating the effects that modifications of tRNAProGGG have on regulating the conformational dynamics of ribosomal complexes in a collaboration with the Hou group at Thomas Jefferson University. The preliminary results from my smFRET experiments suggest that tRNAProGGG modifications do indeed play a role in modulating the dynamics of ribosomal complexes. More importantly, the combination of tRNA without modifications and mRNA carrying a sequence that is prone to induce +1 FS dramatically alters ribosome dynamics, probably by affecting tRNA flexibility, tRNA-ribosome, tRNA-mRNA, and mRNA-ribosome interactions, all of which could have important implications for how +1 FS occurs.

Protein translation has emerged as an important mediator of cellular activity. Here, we discuss efforts to develop and apply new technologies designed to gain insights into translational control. We begin with the application of ribosome profiling to a RiboTag Glioma mouse model which enables translational profiling of transformed cellular populations. This approach demonstrates a number of abnormalities of translation in transformed cells. We go on to report the development of an inexpensive and rapid library preparation methodology which enables high-throughput sequencing of ribosome-protected footprints from small amounts of input material. We apply this technique to a CAMKII RiboTag mouse model to make new insights into cell-type specific translation. Finally, we describe efforts to investigate translation regulatory networks through the development of a technique which couples large-scale perturbation with a genome-wide readout of translation. Molecular dissection of tissues through the ectopic expression of modified ribosomal proteins commonly relies on tissue-specific genes which act as drivers. In the case of glioma, a gene specific to transformed tissue, but not expressed in normal brain tissue, has not been identified. Chapter 2 focuses on efforts to bypass this through the development of a RiboTag Glioma mouse model which allows for concurrent transformation and the expression of an epitope-tagged ribosomal protein in virally infected cells. This model made possible the isolation of translating mRNA from transformed cellular populations and was used to demonstrate the existence of a number of translational abnormalities in transformed cells. Conventional ribosome profiling is a powerful tool which allows for the identification of ribosome-protected mRNA footprints. However, it is time-consuming, expensive, and difficult to implement. Based on our experiences with conventional ribosome profiling, we sought to develop a method which could decrease the overall number of enzymatic reactions and purification steps, thereby reducing the time and cost associated with the procedure; these efforts are discussed in Chapter 3. Utilizing a ligation-free library preparation process, which incorporates poly(A)-polymerase, template switching and bead-based purification, we reduced the time, costs and input requirements required to generate a ribosome profiling library while maintaining high library complexity. We applied our ligation-free ribosome profiling technique to a CAMKII RiboTag mouse model which enabled us to identify patterns of cell-type specific translation and the effects of mTOR inhibition in CAMKII-expressing excitatory neurons. Regulation of protein expression is an essential and highly complex cellular activity. Aberrations of translational control are central to a host of pathologies and have direct clinical relevance. However, our knowledge of the networks which control translation is limited. Chapter 4 details our efforts to develop a highly-scalable technology which enables the identification of gene-specific translational alteration in response to perturbation. Coupled with a large-scale perturbation screen, this technique could lead to the generation of a network for translational control, similar to efforts previously undertaken to understand transcriptional control. By combining the recently developed PLATE-Seq method, which utilizes unique barcode identifiers and pooled library construction, with a technique for the identification and isolation of ribosome associated mRNA, we are able to rapidly and inexpensively determine genome-wide translational states in a highly scalable

Ph.D. / Molecular Biology / Neurospora crassa arg-2 gene encodes the small subunit of arginine specific carbamoyl phosphate synthethase, the first enzyme in fungal arginine (Arg) biosynthesis. The arg-2 mRNA contains an upstream open reading frame (uORF) specifying an evolutionarily conserved 24-residue peptide called the arginine attenuator peptide (AAP). Synthesis of the AAP causes ribosomes to stall on the mRNA in the presence of high concentrations of Arg. The amino acid sequence of the AAP, and not the sequence of its coding region, is responsible for this regulation. Scanning mutagenesis within the evolutionarily conserved region revealed that some residues are more important than others for the AAP to function. While most known nascent peptides that regulate translation are found encoded as uORFs or as N-terminal leader peptides, the AAP can exert regulatory function whether placed near the N-terminus or internally within a large polypeptide. The AAP’s peptide sensing features are conserved due to regulated stalling of fungal, plant, and animal ribosomes in response to Arg. That the AAP functions as an internal domain to regulate elongation in response to Arg establishes that such domains can provide a means of controlling translational elongation. The minimal sequences required for AAP to function as an internal domain was revealed by systematic deletion of its natural N- and C-terminal regions. Comparative analysis of the AAP with other fungi showed that the evolutionarily conserved region of the peptide is required for regulation. This minimal domain functions when placed as a uORF as seen by toeprint assay. Analyses of Arg analogs provided key insights in the structural requirements for Arg’s role in regulation. These results taken together provide a detailed picture of the requirements for Arg-specific regulation mediated by the AAP.