The RNA polymerase genes rpoBC1C2 (the rpoB and rpoC2 are incomplete) of the cyanobacterium Nostoc commune have been cloned, sequenced, and expressed both in vivo and in vitro using E. coli systems. The rpo genes encode large subunits of DNA-dependent RNA polymerase. Two genes in N. commune, rpoC1 and rpoC2, correspond to rpoC of E. coli, which indicates a divergent evolution of RNA polymerase. The rpoBC1C2genes of Nostoc are linked in the order of rpoB, rpoC1, and rpoC2, and are transcribed differently from the corresponding rpo genes of E. coli. In E. coli the rpoBC genes are co-transcribed, together with two ribosomal protein genes. The Nostoc rpoB gene is transcribed by one promoter while the rpoC1C2 genes are co-transcribed by another promoter. Northern analysis of Nostoc RNA revealed two transcripts (3.1 and 5.6 kb), which were specific for the rpoB and rpoC1C2 genes, respectively. SDS-PAGE, Coomassie staining and immunoanalysis detected two polypeptides with molecular weights of 72 and 94 kd when the cloned rpoBC1C2 fragment was expressed in E. coli systems. These two polypeptides were assigned as products of rpoC1 and rpC2, respectively. The transcription of RNA polymerase genes of N. commune is sensitive to water stress (drying). The rpo transcription ceases upon drying and resumes after the dried cells have been rewetted for more than 5 min. The RNA polymerase enzyme itself, however, is stable under the same drying conditions. / Ph. D.

The Nrd1-Nab3-Sen1 (NNS) complex carries out the RNA polymerase II (Pol II) transcription termination of non-coding RNAs (ncRNAs) in yeast, although the detailed interactions among its subunits remain obscure. In this dissertation, we have identified three sequence motifs in Sen1 that mediate direct interactions with the Pol II CTD interaction domain (CID) of Nrd1, determined the crystal structures of these Nrd1 interaction motifs (NIMs) bound to the CID, which elucidated the molecular basis for their recognition by Nrd1 CID, and characterized the interactions in vitro and in yeast. Although the Sen1 NIMs are not essential for supporting viability from the in vivo studies, termination defects were observed from NIM deletions in a reporter assay. In addition, the conservation of Sen1 NIMs suggests these interactions are very likely to promote NNS function. This dissertation also describes the structural studies of the flowering time control protein FPA in plants, which regulates the alternative 3’-end processing of the FLOWERING LOCUS C (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3’-end formation or the functions of the FPA SPOC domain cannot be disrupted by this combination of mutations.

RNA polymerase II (RNAP II) transcribes essentially all messenger RNAs (mRNAs) in eukaryotes. The C-terminal domain (CTD) of its largest subunit contains consensus heptad repeats Y₁S₂P₃T₄S₅P₆S₇. Dynamic post-translational modifications of the CTD regulate RNAP II transcriptional activity and also facilitate transcription-coupled RNA processing events. One important mark is phosphorylation at Ser5 position, whose level peaks during transcription initiation but gradually diminishes toward the 3' end of genes. Ssu72 is a known CTD pSer5 phosphatase. Recent studies identified a binding partner of Ssu72, symplekin, which is an essential scaffold protein in pre-mRNA 3' end processing. Little is known about the molecular function of symplekin and neither do we understand how the symplekin-Ssu72 interaction couples pre-mRNA 3' processing to transcription. We first determined the crystal structure of the symplekin-Ssu72-CTD phosphopeptide complex. The N-terminal domain of symplekin embraces Ssu72 with its HEAT-repeat motif, serving as a typical molecular scaffold. Strikingly, the CTD phosphopeptide bound to the active site of Ssu72 has the peptide bond between pSer5 and Pro6 in the cis configuration, distinct from all known CTD conformations, which were exclusively in trans. While it was generally believed that only the trans peptide bond is recognized by proline-directed serine/threonine phosphatases or kinases, our discovery demonstrates for the first time that Ssu72 targets the energetically less-favorable cis peptide bond. In addition, we found that the binding of symplekin and also the presence of a proline cis-trans isomerase can stimulate the phosphatase activity of Ssu72 in vitro. The symplekin-Ssu72 interaction as well as the catalytic activity of Ssu72 is required in our transcription-coupled polyadenylation assay. Overall, our study has important implications for the regulation of RNAP II transcription by cis-trans isomerization of the CTD and will help us understand how CTD modifications influence the recruitment of pre-mRNA 3' end processing factors in a transcription-coupled manner. Recent studies showed that Ssu72 is also a phosphatase of CTD pSer7, which is involved in small nuclear RNA transcription and 3' end processing. However, a pSer7 phosphatase activity appears to be inconsistent with our structure because pSer7 is followed by Tyr1' of the next repeat rather than a proline, and it is unlikely for the pSer7-Tyr1' peptide bond to be in cis configuration. To solve this conundrum, we determined the crystal structure of the pSer7 CTD peptide bound to Ssu72. Surprisingly, the backbone of the pSer7 CTD runs in an opposite direction compared with the pSer5 CTD, allowing a trans pSer7-Pro6 peptide bond to be accommodated in the active site. However, Ssu72 has a much lower affinity for pSer7 than pSer5 and several structural features are detrimental for the catalytic activity towards pSer7. Consistent with these observations, our in vitro assays showed that the dephosphorylation of pSer7 by Ssu72 is ~4000-fold lower than that of pSer5. This further characterization of Ssu72 not only presents the first phosphatase in the literature that recognizes peptide substrates in both directions but also provides a more comprehensive understanding on CTD regulation by phosphatases from a structural perspective. Another protein, Rtr1, was recently suggested to function as a pSer5 phosphatase in a zinc-dependent fashion, separately or redundantly with Ssu72. We solved the crystal structure of Rtr1 and discovered a new type of zinc finger with no close structural homologs. Unexpectedly, Rtr1 does not present any evidence of an active site and it lacks detectable phosphatase activity in all our assays. We believe that, based on our results, Rtr1 does not have catalytic ability but instead indirectly regulate the phosphorylation state of the CTD. In summary, our studies on the symplein-Ssu72-CTD complex as well as Rtr1 have revealed several novel structural features that are essential for the CTD regulation at the atomic level. These results will also shed light on understanding the mechanism by which RNAP II transcription and RNA processing are coupled.

Hepatitis C virus (HCV) replication is directed by NS5b, the viral RNA dependent RNA polymerase (RdRp). To date, our understanding of the HCV polymerase has come almost entirely from genotype 1. The aim of this study was to examine the influence of sequence variation in the polymerase region by characterising a polymerase derived from genotype 3a. The genotype 3a CB strain polymerase was cloned into the bacterial expression vector pTrcHis2C incorporating a hexahistidine tag to facilitate purification. An optimised process produced 2.5 mg of highly purified recombinant protein per litre of bacterial culture. The 3a preparation possessed an RdRp activity and could utilise both homopolymeric and heteropolymeric RNA templates. Optimal activity was seen at 30oC at pH 8 in reactions containing 160nM enzyme, 10??g/ml RNA template and 2.5mM MnCl2. Subsequently, three genotype 1b polymerases including the HCV-A, Con1 and JK1 strains were cloned for the comparison of activity under identical conditions. Steady state kinetic parameters for GMP incorporation revealed the 3a polymerase exhibited the highest activity, with an almost two fold higher catalytic efficiency (Kcat/Km) than HCVA-1b, primarily due to differences in Km for GTP (2.984??M vs 5.134??M). Furthermore, the 3a polymerase was 3.5 fold and 15 fold more active than JK1-1b and Con1-1b respectively. Improving our understanding of the influence of sequence difference on polymerase activity, particularly in the context of replication will be crucial to developing effective antiviral therapies.

The aim of this study was to gain more information about the interactions between DNA and the histone octamer during the process of transcription. This work used a pUC8 plasmid derivative that contained the core promoter region of the RNA polymerase I of Acanthamoeba castellanii, placed upstream of four repeats of the 5S rDNA nucleosome positioning sequence from the sea urchin, Lytechinus variegatus. The plasmid was reconstituted into chromatin via addition of chicken erythrocyte histone octamers, using polyglutamic acid as a nucleosome assembly factor. The positioning of nucleosomes on the insert was monitored by restriction enzyme digestion. Proper nucleosome positioning was shown to be dependent on the presence of preassembled transcription complexes on the promoter region. The absence of preformed transcription complexes on the promoter region prior to nucleosome reconstitution perturbed the distribution of histone octamers on the repeats of the 5S rDNA. This "mispositioning" effect was related to the location of the RNA polymerase I promoter region upstream of the four repeats of the 5S rDNA fragment. Band shift assays in polyacrylamide gel electrophoresis were used to determine the relative efficiency of nucleosome formation on the promoter-containing fragment, on 5S rDNA and finally on nucleosome core particle DNA. The results indicate that the promoter fragment forms a nucleoprotein complex at lower concentration of histone than the 5S positioning sequence. This complex may not be a nucleosomal structure. The reconstituted plasmid was then used to investigate the transcriptional elongation by RNA polymerase I using the chromatin-like template containing positioned nucleosomes as compared to transcription on improperly positioned nucleosomes and on free DNA. The efficiency of transcription was related to the proper positioning of nucleosomes with regard to the tandemly repeated 208-bp 5S rDNA. The presence of phased nucleosomes in the path of the transcription complex seemed not to inhibit nor to significantly slow down the elongation as compared to free DNA. Furthermore, nucleosome positioning, as assayed by restriction endonuclease digestion, did not change after passage of the polymerase I transcription complex. / Graduation date: 1993

Thesis (Ph. D.)--Rutgers University, 2008. / "Graduate Program in Chemistry and Chemical Biology." Includes bibliographical references (p. 133-142).

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Chemistry." Includes bibliographical references (p. 147-163).

Hepatitis C virus (HCV) replication is directed by NS5b, the viral RNA dependent RNA polymerase (RdRp). To date, our understanding of the HCV polymerase has come almost entirely from genotype 1. The aim of this study was to examine the influence of sequence variation in the polymerase region by characterising a polymerase derived from genotype 3a. The genotype 3a CB strain polymerase was cloned into the bacterial expression vector pTrcHis2C incorporating a hexahistidine tag to facilitate purification. An optimised process produced 2.5 mg of highly purified recombinant protein per litre of bacterial culture. The 3a preparation possessed an RdRp activity and could utilise both homopolymeric and heteropolymeric RNA templates. Optimal activity was seen at 30oC at pH 8 in reactions containing 160nM enzyme, 10??g/ml RNA template and 2.5mM MnCl2. Subsequently, three genotype 1b polymerases including the HCV-A, Con1 and JK1 strains were cloned for the comparison of activity under identical conditions. Steady state kinetic parameters for GMP incorporation revealed the 3a polymerase exhibited the highest activity, with an almost two fold higher catalytic efficiency (Kcat/Km) than HCVA-1b, primarily due to differences in Km for GTP (2.984??M vs 5.134??M). Furthermore, the 3a polymerase was 3.5 fold and 15 fold more active than JK1-1b and Con1-1b respectively. Improving our understanding of the influence of sequence difference on polymerase activity, particularly in the context of replication will be crucial to developing effective antiviral therapies.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 80-87). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1981.