Protein synthesis, or translation, is a complex, multi-step process that requires regulatory and quality control mechanisms to ensure the accurate production of proteins. Two major challenges during bacterial protein synthesis are maintaining the accuracy of translation during the elongation stage and resolving stalled ribosomal complexes. Interestingly, bacteria have evolved two mechanisms, a post-peptidyl transfer quality control (post PT QC) and a ribosome rescue mechanism, to counter these challenges. Both of these mechanisms make use of a protein factor that normally functions during translation termination, Release Factor 2 (RF2), along with an additional protein factor, Release Factor 3 (RF3) for post PT QC and Alternative ribosome-rescue factor A (ArfA) for ribosome rescue, to achieve these non-canonical functions. The mechanistic role of RF3 and ArfA in these two pathways remains unclear; however, they may play a role in regulating RF2 in context of these non-canonical pathways. As a step toward understanding the role of RF3 and ArfA in post PT QC and ribosome rescue and, in particular, their role in the regulation of RF2, I sought to determine the effect of RF3 and ArfA on the binding kinetics of RF2 in post PT QC and ribosome rescue pathways. Using a single-molecule fluorescence resonance energy transfer (smFRET) signal between the P-site peptidyl-tRNA and RF2, the binding and dissociation of RF2 can be directly monitored in the absence or in the presence of RF3 or ArfA. In Chapter 2, I describe the development of smFRET signals using different chromophores, cyanine 3 to cyanine 5 (Cy3-Cy5) or to a fluorescence quencher (Cy3-QSY9). The Cy3-Cy5 and Cy3-QSY9 smFRET signals complement each other for monitoring RF2 binding; whereas Cy3-Cy5 is suitable for observing stable binding using low substrate concentration, Cy3-QSY9 is suitable for observing transient binding using high substrate concentration. The RF2 binding and dissociation to ribosomal complexes was first examined in the absence of other factors thus providing the foundation for studying the regulation of RF2 binding by RF3 or ArfA. In the bacterial post PT QC mechanism, RF3 enhances the rate of RF2-mediated peptide release to catalyze premature termination of miscoded protein, thus ultimately increasing the fidelity of protein synthesis1. Without addition of RF3, the rate of RF2-mediated peptide release is too slow to compete with the rate of protein synthesis. In Chapter 3, the role of RF3 on RF2 binding kinetics in post PT QC was investigated using both an fMet-Lys-tRNALys(Cy3) to RF2(Cy5) smFRET signal and an fMet-Lys-tRNALys(Cy3) to RF2(QSY9) smFRET signal. The ArfA-RF2 ribosome rescue pathway is a backup mechanism for trans-translation, which relieves stalled ribosomal complexes by providing an open reading frame coding for both a degradation tag and a stop codon2. Because the expression of ArfA is under strict control by trans-translation, the ArfA-RF2 pathway only functions in the absence of active trans-translation. More importantly, deletion of both the trans-translation and ArfA-RF2 pathways leads to synthetic lethality in E. coli, highlighting the critical role of ribosome rescue in vivo3. In Chapter 4, I used an fMet-Phe-tRNAPhe(Cy3) to RF2(Cy5) smFRET signal to evaluate the role of ArfA on RF2 binding and dissociation in the ribosome rescue pathway. Collectively, these studies survey the regulation of RF2 binding kinetics by RF3 or ArfA in performing non-canonical functions such as post PT QC and ribosome rescue in bacteria.

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination and post-germination. Here I test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating and post-germination seeds. It is demonstrated that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean seed germination and post-germination. Immunogold electron microscopy (EM) with VSR antibodies demonstrates that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in Day-1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at Day-3 post-germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating and post-germination seeds. Further immunogold EM studies demonstrate that VSR and aleurain colocalize to MVBs, as well as PSVs in germinating and post-germination seeds. Thus, MVBs in germinating and post-germination seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed, and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination and post-germination. / Storage proteins synthesized during seed development are transported to PSVs for storage. However, relatively little is known about the mechanisms of storage protein transport. A putative VSR-interacting protein termed S2 was identified as mung bean 8S globulin. Thus, I test the hypothesis that VSR proteins may be involved in storage protein transport to PSVs in developing mung bean seeds. Immunogold EM with 52 (8S globulin) antibody demonstrates that transport of 8S globulin to PSVs is Golgi-mediated, involving dense vesicle (DV) and a novel prevacuolar compartment (PVC). The novel PVC consists of storage protein aggregates and small internal vesicles. Immunogold EM with S2 (8S globulin) antibody demonstrates that MVBs contain 8S globulin at early stage of seed development. Further immunogold EM studies demonstrate that VSR and 8S globulin colocalize to DVs and the novel PVCs. In vitro binding study demonstrates that calcium ion can stabilize interaction between VSRs and 8S globulin. Thus, VSR proteins may mediate storage protein transport to PSVs via a novel PVC. / Wang, Junqi. / "March 2007." / Adviser: Jiang Liwen. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0052. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 120-131). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

By determining the Calpha and Cbeta chemical shift of P2 and its relaxation properties, together with secondary structure prediction, P2 was found to have an N-terminal 4-helices structural domain and a C-terminal unstructured coil. / P2 was found to interact with P1, forming heterodimer and with P2, forming homodimer. It was found that dimerization is carried out by their N-terminal, forming NTD-P1/NTD-P2 heterodimer and NTD-P2 homodimer. / Ribosome is the organelle responsible for protein synthesis and it was suggested that P-proteins located at the lateral stalk are involved in this process. Until now, structure of any P-protein is still not known although crystal structure of ribosome was solved. In order to know more about the biological role of P-proteins, structural characterization was carried out on human ribosomal protein P2. / The C-terminal tail which is conserved among P0, P1 and P2 of various species was found to interact with ribosome inactivating protein (TCS). This helps delivering TCS to its RNA substrate and carrying out its N-glycosidase activity. It was also found that TCS and EF2 are close in space suggesting that binding of TCS to P-proteins may hinder the association of EF2 to P-protein, thus inhibiting protein translation. / The solution structure of NTD-P2 homodimer was solved. It has 8 helices, 4 from each monomer. The surface is hydrophilic and the core is hydrophobic with a hydrophobic dimeric interface. By circular dicroism measurement, structural alignment and secondary structure prediction, we hypothesize that the dimerization mode of NTD-P1/NTD-P2 heterocomplex should be similar to NTD-P2 homodimer. Therefore, homology modeling was used to model the structure of NTD-P1/NTD-P2 using NTD-P2 as template. Interestingly, there is a small exposed hydrophobic patch on NTD-P1 which is lack in NTD-P2. This exposed hydrophobic patch may be the potential P0 binding site, forming P0(P1/P2)2 complex. Moreover, this exposed hydrophobic pocket is smaller than that of prokaryotic counterpart, thus providing insight in ribosome assembly and regulation in protein translation. / Lee, Ka Ming. / Advisers: K. B. Wong; P. C. Shaw. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0096. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 121-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

by Tsang Kwok Yeung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 82-93). / ACKNOWLEDGMENTS --- p.I / ABSTRACT --- p.II / TABLE OF CONTENTS --- p.V / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- PURIFICATION AND CHARACTERIZATION OF A TYPE I RIP MOMORGROSVIN FROM THE SEEDS OF MOMORDICA GROSVENORI --- p.34 / GENERAL DISCUSSION --- p.80 / REFERENCES --- p.82

Includes bibliography.

Thesis (Ph. D.)--Pennsylvania State University, 1973. / Includes bibliographical references.

Graduation date: 1996

Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, May 2007. / Title from title screen. Includes bibliographical references.

Although a long standing convention maintained that G Protein Coupled Receptors (GPCRs) exist in the plasma membrane solely as monomers, substantial work over the last two decades has demonstrated that these ubiquitous receptors can and in many cases, preferentially, exist as homodimers, heterodimers, or higher order oligomers. Often, two GPCRs of the same class heterodimerize; it is less common for two GPCRs of different signaling pathways to interact. The work presented here studied the physical and functional interaction of two GPCRs from discrete classes, the Beta 2 Adrenergic Receptor (β2AR), a Gαs-coupled receptor, and Bradykinin type 2 Receptor (Bk2R), a Gαq coupled receptor. These data show that Bk2R and β2AR are physically coupled when heterologously expressed in Xenopus oocytes, and in pheochromocytoma (PC12) cells and in freshly isolated murine ventricular myocytes, two systems that endogenously express these receptors. This physical coupling led to functional consequences in heterologous and endogenous expression systems, as Bk2R was able to transactivate β2AR signaling via its direct interaction with the receptor. Furthermore, coexpression of Bk2R shifted the dose response curve of β2AR for its selective agonist rightward in Xenopus oocyte electrophysiology experiments, suggesting the presence of Bk2R negatively affected β2AR native pharmacology. Up to thirty minutes of either bradykinin (BK) or isoproterenol exposure did not change the relative amount of Bk2R/β2AR heterodimer in PC12 cells, a rat adrenal medulla tumor cell line that endogenously expresses these receptors. Despite the obvious signaling consequences, the Bk2R/β2AR heterodimer accounted for only 10% of the total β2AR protein detected and 20% of the total Bk2R protein detected. When other Bk2R-specific ligands were also tested to examine the extent of β2AR transactivation, our data showed that both Lys-des-Arg-Bradykinin, a Bk2R partial agonist and NPC 567, a Bk2R antagonist, transactivated β2AR to the same extent as BK. Taken together, our data provide a novel mode of receptor regulation and signaling via Bk2R/β2AR heterodimerization. Because a large percentage of therapeutics target GPCRs, a greater understanding of how a GPCR heterodimer functions could be beneficial for targeting new drugs and refining existing drugs. Understanding the Bk2R/β2AR heterodimer provides a new perspective on the myriad of fucntional consequences that occur when a GPCR is activated.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 165-172).