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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.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

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

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / Typescript. Vita. Includes bibliographical references (leaves: 108-124). Also available on the Internet.

Thesis (Ph. D.)--University of Oregon, 1999. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 90-101). Also available for download via the World Wide Web; free to University of Oregon users. Address: http://wwwlib.umi.com/cr/uoregon/fullcit?p9947978.

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;

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 159-176).

The anti-tumor activity of platinum based drugs is mediated by their ability to attack DNA. Platinum complexes can alter the structure of DNA by modifying the bases, mainly guanines. The biological consequnces of such interactions are compromising replication and transcription. RNA polymerase complex can stall at a damaged site in DNA and mark the lesion for repair by proteins that are utilized to execute nucleotide excision repair, a pathway commonly associated with the removal of bulky DNA damage from the genome. This RNA polymerase-induced repair pathway is called transcription-coupled nucleotide excision repair. Main goal of this thesis was to study RNA polymerases tolerance of DNA damage by novel, biologically active platinum (II) complexes involving derivatives of aromatic cytokinines as the ligands; cis-[Pt(2-chloro-6-(4-methoxybenzylamino)-9-isopropylpurin)2Cl2](PR-001), cis-[Pt(2-chloro-6-(benzylamino)-9-isopropylpurin)2Cl2](PR-002 )and cis-[Pt(2-(3-hydroxypropylamino)-6-(benzylamino)-9-isopropylpurin)2Cl2](PR-005). DNA templates (constructs) that contain a single, site-specific DNA lesion and support transcription by human RNA polymerase II and bacteriophage T7 RNA polymerase were prepared. The method is making use of polymerase chain reaction (PCR) and biotin-streptavidin interactions and paramagnetic particles to purify the final product. Synthetic oligomers duplexes (75-mer, 56-mer and 15-mer) are ligated to 5´-biotin pCI-neo-G-lessT7 PCR fragment, the 15-mer is either unmodified or modified with a site-specific lesion of PR-005 and cisplatin. We also studied the inhibition of RNA polymerases activity on globally modified plasmid pCI-neo and pUC 19 by novel platinum complexes and cisplatin. We found that bifunctional adducts of complex PR-005 contrary to adducts of PR-001 and PR-002 effectively decrease amount of full lenght transcripts produced by both, human and bacterial RNA polymerases. This result can be explained by a sterical block, induced to DNA by intrastrand cross-link of PR-005 with bulky aromatic ligands.

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.