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.

Includes bibliography / v, 147 leaves, [31] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1987

"September, 2002." / Bibliography: leaves 93-106. / 106 leaves : ill. (some col.), plates ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / In this thesis the biochemical and genetic characterization of Fkh2p identifies it as a major component of SFF. It has been shown to bind DNA in an Mcm1p dependent manner and the Fkh2p DNA binding domain is essential for this interaction. The protein interaction domain of Mcm1p has been demonstrated to be essential for ternary complex formation. Fkh2p, along with a functionally redundant protein Fkh1p, has been show to control the periodic expression of the CLB2 cluster genes. The functional characterisation of the Fkh2p domains reveals an important role for both the Forkhead associated domain and the C-terminus. Ndd1p. another protein important for mitotic progression, is shown to be important for CLB2 cluster regulation by de-repressing Fkh2p and activating gene expression. The role of cdk activity is shown to act through the CLB2 cluster upstream activating sequences, possibly through Ndd1p. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2003

Two novel and highly related C₂H₂ zinc finger proteins (CTIP1/BCL11A/EVI9 and CTIP2/BCL11B/Rit1) have been implicated in COUP-TF signaling, etiology of myeloid and lymphoid malignancies, and hematopoietic cell development. However, the precise cellular function(s) and the contribution of these proteins to neoplastic processes and hematopoietic cell development remain unknown. The goal of the studies described herein was to elucidate the molecular mechanisms underlying the transcriptional repression mediated by these proteins to understand their biological properties, and ultimately, their cellular function(s). CTIP proteins repressed transcription of a reporter gene in a TSA-insensitive manner, suggesting that this repression mechanism(s) may not involve TSA-sensitive histone deacetylation catalyzed by member(s) of class I and II HDACs. One possible mechanism is that CTIP proteins may exert ISA-insensitive histone deacetylation catalyzed by TSA-insensitive HDAC(s), such as SIRT1, to repress transcription. In deed, SIRT1 was found to interact with CTIP proteins both in vitro and in mammalian cells, and was recruited to the promoter template in a CTIP-dependent manner. The proline-rich regions of CTIP proteins and the sirtuin homology domain of SIRT1 were found to be essential for mediating CTIPs•SIRT1 interactions. Moreover, column chromatography revealed that SIRT1 and CTIP2 were components of a large complex in Jurkat cell nuclear extracts. Based on the findings that SIRT1 associates with CTIP proteins in mammalian cells, SIRT1 may underlie the transcriptional repression activity of CTIP proteins. The following results support the hypothesis that SIRT1 may underlie the mechanism(s) of CTIP-mediated transcriptional repression. First, CTIP-mediated transcriptional repression was inhibited, at least partially, by nicotinamide, an inhibitor of the NAD⁺-dependent, TSA-insensitive HDACs. Second, the decrease in levels of acetylated histones H3 and/or H4 at the promoter region of a reporter gene was observed upon overexpression of CTIP proteins, and this effect was inhibited, at least partially, by nicotinamide. Third, endogenous SIRT1 was recruited to the promoter template of a reporter gene in mammalian cells upon overexpression of CTIP proteins. Fourth, SIRT1 enhanced the transcriptional repression mediated by CTIP proteins and this enhancement required the catalytic activity of SIRT1. Finally, SIRT1 enhanced the deacetylation of template-associated histones H3 and/or H4 in CTIP-transfected cells. In summary, results described herein strongly suggest that CTIP-mediated transcriptional repression involves the recruitment of SIRT1 to the template, at which the TSA-insensitive, but nicotinamide-sensitive histone deacetylase catalyzes deacetylation of promoter-associated histones H3 and/or H4. These results contribute additional understanding to the molecular mechanisms underlying transcriptional activity of CTIP proteins, which might be helpful for identification and characterization of the target genes under the control of CTIP proteins in cells of hematopoietic system and/or the central nervous system. / Graduation date: 2004

Thesis (Ph. D.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

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

Polypurine·polypyridine (pur·pyr) tracts are a run of all purines on one strand and all pyrimidines on the complementary DNA strand. Statistical overrepresentation of the tracts in eukarocytes suggests a cellular role or roles. The tracts from triplex DNA <i>in vitro</i> and there is evidence for triplex DNA <i>in vivo</i>. Several cellular roles are possible for triplex DNA. The presence of the tracts in gene 5' flanking regions suggets a regulatory role. This work investigates the role of triplex DNA in the cell, particularly in the regulation of transcription. Proteins mediate DNA looping in the regulation of transcription and in its condensation in chromosomes. Such looping may also be mediated by transmolecular triplexes, formed between separated pur·pyr tracts. Formation of pyr·pur·pyr transmolecular triplexes was investigated using linear and circular plasmid models containing separated pur·pyr tracts able to form a triplex with each other, but not within a tract. Transmolecular triplex loops (T-loops) formed in circular DNA, suggesting a possible regulatory or structural role <i>in vivo</i>. The following model shows a T-loop formed at pH 4. At pH 6, a duplex partially reforms and single-stranded region(s) trap the structure. and single-stranded region(s) trap the structure. T-loops were used as a model to test the Idea that a single-strand extruded by triplex formation in the 5' flanking region of a gene could promote transcription. Transcription was inhibited in T-loops, suggesting such structures could block transcriptional elongation if formed <i>in vivo</i>. The ability of polyamine analogues to promote triplex formation was also tested using T-loops. Pentamines promoted T-loop formation at lower concentrations than tetramines. Spatial distribution of charge was also important. A triplex role in transcriptional regulation was investigated using two examples of human genes with 5' flanking pur·pyr tracts. The effect of triplex-specific antibodies on expression of c-' myc' was investigated using agarose-encapsulated nuclei. Triplex formation between c-'src' promoter pur·pyr tracts was visualized as gel band shift die to dimerization between linear plasmid fragments containing individual tracts. A transmolecular triplex was proposed as one way in which the c-'src' tracts could form a triplex <i>in vivo</i> which might be involved in the regulation of transcription.

Thesis (M.S.)--Auburn University, 2008. / Abstract. Includes bibliographical references (p. 39-42).

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 170-205).

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 103-114). Also available in print.