Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [176]-190).

Thesis (Ph.D. in Molecular Biology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 100-113). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Transcription initiation is a key step at which gene expression can be regulated. The sigma subunit of RNA polymerase provides the enzyme with the ability to recognize promoter sequences and initiate transcription at specific sites on the chromosome. The molecular basis of sigma function is not well known. It has been suggested that sigma factors confer promoter specificty by making direct contacts to the promoter DNA (Losick and Pero, 1981). To test this idea, suppressors of promoter down mutations were sought that affected the promoter recogniton properties of the σ70 subunit of E. coli RNA polymerase. Four such sigma mutants were obtained, two of which are allele-specific. One of these mutants has a change at a position in the predicted helix-turn-helix DNA binding structure which lies in a conserved region of the protein (region 4). This mutant specifically suppresses promoter down mutations in the -35 region of the promoter. The other mutant has a change at a residue that lies in a predicted α-helix of conserved region 2. This mutant specifically suppresses promoter mutations in the -10 region of the promoter. These data support the idea that regions 2 and 4 of sigma interact with the -10 and -35 regions of the promoter, respectively.

The poultry-to-human transmission of the influenza virus and the recent H1Nl influenza pandemic have become major concerns worldwide. The nucleoprotein (NP) of influenza virus binds the RNA genome and plays essential role in transcription and replication during the virus life cycle. / The study leads to a better understanding towards the RNP organization of influenza virus and provides information for the future design of anti-influenza agents. / We have also shown, by RNP reconstitution assay and co-immunoprecipitation, that the interaction between NP and PB2 is crucial for the proper functioning of the RNP. The functional association of NP and PB2 requires either the PB2 host-determining residue lysine-627 or arginine-630 with the latter involving NP arginine-150 also. Using SPR, we have demonstrated that both residues take part in the direct protein-protein interaction, without the involvement of RNA. These results suggest a dual interaction mechanism between NP and PB2. This may confer replication advantages to the virus, as either one can give an active RNP and explains the increased virulence of avian influenza viruses carrying the E627K mutation in mammalian cells. In addition, our findings identify the NP-PB2 interacting surface, with the PB2 627/630 region facing the RNA binding groove of NP. / We have determined the 3.3 A crystal structure of H5N1 NP, which is composed of head and body domains and a tail loop. Using surface plasmon resonance (SPR), we found the basic loop (residues 73-91) and arginine-rich groove, but mostly a protruding element centering at R174 and R175, to be important in RNA binding. Ribonucleoprotein (RNP) reconstitution assay with these multiple-point and deletion mutants indicate their functional importance towards the transcription-replication activities of the virus polymerase. Single-point mutations at these concerned regions do not have a significant effect on their RNP activities, suggesting that NP mediates RNA-binding through multiple residues. / Ng, Ka Leung. / Adviser: Pang Chui Shaw. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 121-136). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

The RNA interference (RNAi) pathway in Caenorhabditis elegans is a two-step, small RNA-mediated silencing pathway. Unlike in other organisms, Dicer processing of double-stranded RNA into small interfering (si) RNAs is not sufficient in worms to induce gene silencing. The activity of cellular RNA-dependent RNA polymerase (RdRP) is necessary to synthesize a secondary pool of siRNAs, which interact with a unique class of Argonaute proteins to form the functional effector complexes that mediate silencing. The aims of this thesis were to: 1) characterize the role of RdRP family members in endogenous small RNA biogenesis; 2) identify the Argonaute proteins that interact with RdRP-dependent small RNAs; and 3) investigate the biological function of RdRP-dependent small RNA pathways in C. elegans. In this thesis, I describe genetic, deep sequencing, and molecular studies, which identify 22G-RNAs as the most abundant class of endogenous small RNA in C. elegans. The 22G-RNAs resemble RdRP-dependent secondary siRNAs produced during exogenous RNAi, in that they possess a triphosphorylated 5’ guanine residue and exhibit a remarkable strand bias at target loci. Indeed, I show that 22G-RNAs are dependent on the activity of the RdRPs RRF-1 and EGO-1 and function in multiple distinct endogenous small RNA pathways. Interestingly, I have found that RRF-1 and EGO-1 function redundantly in the germline to generate 22G-RNAs that are dependent on and interact with members of an expanded family of worm-specific Argonaute (WAGO) proteins. The WAGO/22G-RNA pathway appears to be a transcriptome surveillance pathway that silences coding genes, pseudogenes, transposons, and non-annotated, or cryptic, transcripts. In contrast, I have found that EGO-1 alone is required for the biogenesis of a distinct class of 22G-RNAs that interact with the Argonaute CSR-1. Surprisingly, the CSR-1/22G-RNA pathway does not appear to silence its targets transcripts. Instead, the CSR-1/22G-RNA pathway is essential for the proper assembly of holocentric kinetochores and chromosome segregation. Lastly, I show that a third endogenous small RNA pathway, the ERI pathway, is a two-step silencing pathway that requires the sequential activity of distinct RdRPs and Argonautes. In the first step of this pathway, the RdRP, RRF- 3, is required for the biogenesis of 26G-RNAs that associate with the Argonaute, ERGO-1. In the second step, RRF-1 and EGO-1 generate 22G-RNAs that associate with the WAGO Argonautes. This work demonstrates how several C. elegans small RNAs pathways utilize RdRPs to generate abundant populations of small RNAs. These distinct categories of small RNAs function together with specific Argonaute proteins to affect gene expression, to play essential roles in development, and in the maintenance of genome and transcriptome integrity.