<p>The cytoplasmic ring (C-ring) of the flagellar motor consists of three proteins: FliG, FliM, and FliN, each present in different copy numbers. These proteins perform the function of transmitting torque from the stators to the basal body, as well as regulating the rotational direction of the flagellum. Despite decades of study and great progress towards the understanding of the molecular details of the flagellum&rsquo;s mode of action, substantial questions still remain about its detailed architecture and molecular mechanisms. Here we describe a series of in vitro and in vivo experiments designed to provide insight into the structure of the flagellar C-ring. </p><p> We begin this work by presenting a background of the forms of cellular motility and provide context for the flagellum within the great diversity of motility mechanisms. We then summarize the bacterial chemotaxis signal transduction system, one of the most deeply characterized signaling pathways within biology. Lastly we introduce the flagellum with a focus on C-ring structure and function. </p><p> The research portion begins with a characterization of the interaction between the FliG N-terminal domain (FliG_N) and the C-terminal region of the flagellar membrane protein FliF (FliF_C). We find that these two proteins interact strongly and that this interaction causes widespread conformational changes throughout FliG_N. Based on NMR and other biophysical data we propose a binding site for FliF_C centered on helix 1 of FliG_N. </p><p> In the next section we further characterize the interaction between FliF_C and FliG_N. We generate a fusion FliF_C-FliG_N polypeptide and characterize this complex. Using spin labeling experiments we confirm our predicted interaction site between FliF_C and FliG_N. We also identify a novel interaction between an important hydrophobic patch on the FliG_NM linker and FliG_N. </p><p> Next we study and characterize the domain architecture of the full-length FliG_NMC protein. By evaluating pair-wise domain interactions and comparing NMR spectra of numerous FliG constructs, in combination with in vivo experiments, we provide evidence that the FliG middle- (FliG_M) and C-terminal (FliG_C) domains interact in an intra-protomer manner. This model is in excellent accord with the 3D structure of the <i>Salmonella typhimurium</i> C-ring as derived from cryo-EM. </p><p> Lastly, we describe a number of experiments probing complex formation between FliG, FliM and FliN, with the aim of determining how these interactions are modulated by CheY binding. </p>

<p> In the D1-D61N mutant, it was possible to resolve a clear lag phase prior to the appearance of O2, indicating formation of an intermediate before onset of O2 formation. The lag phase and the photochemical miss factor were more sensitive to isotope substitution in the mutant indicating that proton efflux in the mutant proceeds via an alternative pathway. The results are discussed in comparison with earlier results obtained from the substitution of CP43-Arg357 with lysine and in regards to hypotheses concerning the nature of the final steps in photosynthetic water oxidation. These considerations lead to the conclusion that proton expulsion during the initial phase of the S3-S0 transition starts with the deprotonation of primary catalytic base, probably CP43-Arg357, followed by efficient proton egress involving the carboxyl group of D1-D61 in a process that constitutes the lag phase immediately prior to O2 formation chemistry. The asparagine, phenylalanine and threonine substitutions to D1-V185 were able to accumulate significant levels of charge separating PSII. Of the three substitutions the phenylalanine substitution was the most severe with a complete inability to evolve oxygen, despite being able to accumulate Photosystem II and to undergo stable charge separations. The threonine substitution had no apparent effect on oxygen evolution other than a 40% reduction in the steady state rate of O2 production compared to type Synechocystis, which can be attributed to that mutants reduced ability to accumulate PSII. The asparagine substitution produced the most complex phenotype. While still able to evolve oxygen, it does so less efficiently than wild type PSII, with a miss factor 4% higher than wild type Synechocystis. The substitution on D1-Val185 with asparagine also decreased the t1/2 of O2 release from thylakoid membranes from 1.2 ms to 10.0 ms and decreased the t1/2 lag phase prior to the onset of O2 release to 2.8 ms. The combination of a long lag period and a decreased rate of O2 release can also be observed in the D1-D61N mutant strains of Synechocystis and in PSII centers in which chloride has been replaced by iodide.</p>

<p> Dicer plays a central role in RNA interference pathways by cleaving double-stranded RNAs (dsRNAs) to produce small regulatory RNAs. Human Dicer can process long double-stranded and hairpin precursor RNAs to yield short interfering RNAs (siRNAs) or microRNAs (miRNAs), respectively. In humans, Argonaute2 (AGO2) assembles with the guide RNA-generating enzyme Dicer and either the RNA-binding protein TRBP or PACT to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs from Dicer to AGO2. Here, I have used electron microscopy and single particle analysis of human Dicer-RNA complexes and the RLC to gain insight into the structural basis for human Dicer's substrate preference and RISC-loading. My studies show that Dicer traps pre-siRNAs in a non-productive conformation, while interactions of Dicer with pre-miRNAs and dsRNA binding proteins induce structural changes in the enzyme that enable productive substrate recognition in the central catalytic channel. The RLC Dicer's N-terminal DExH/D domain, located in a short base branch, interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2.</p>

Thesis (Ph. D.)--University of Connecticut, 2006. / (UnM)AAI3205761. Adviser: Glenn King. Source: Dissertation Abstracts International, Volume: 67-02, Section: B, page: 0878.

Thesis (Ph. D.)--University of Illinois at Urbana-Champaign, 2006. / (UMI)AAI3223565. Adviser: Huimin Zhao. Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3588.

Thesis (Ph. D.)--University of California, San Francisco, 2008. / Source: Dissertation Abstracts International, Volume: 68-11, Section: B, page: 7314. Adviser: Kevan M. Shokat.

Thesis (Ph. D.)--University of California, San Francisco, 2006. / Source: Dissertation Abstracts International, Volume: 67-05, Section: B, page: 2415. Adviser: Robert J. Fletterick.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3662. Adviser: Huimin Zhao. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Thesis (Ph.D.)--University of California, San Francisco, 2010. / Source: Dissertation Abstracts International, Volume: 71-02, Section: B, page: . Adviser: John D. Gross.

Thesis (M.S.)--Fairleigh Dickinson University, 1983. / Source: Masters Abstracts International, Volume: 35-05, page: 1318.