published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

In order to expand our understanding of the expression of chloroplast genes, the structure and expression of the Euglena gracilis rpoC1 and rpoC2 loci were studied. The rpoC1 and rpoC2 gene products are similar to the amino- and carboxyl-terminal regions of the $\beta\sp\prime$ subunit of E. coli RNA polymerase. The nucleotide sequence (7,270 bp) was determined for 100% of both strands encoding these two genes. The rpoC1 and rpoC2 genes are located downstream and in the same polarity as the rpoB gene. The organization of the Euglena rpoB-rpoC1-rpoC2 genes is conserved in plant chloroplasts and is similar to the E. coli rpoB-rpoC operon. The Euglena rpoC1 gene (586 codons) encodes a polypeptide with a predicted molecular weight of 68,043. The rpoC1 gene is interrupted by one group II intron of 349 bp, seven group III introns of 107, 100, 119, 97, 110, 102 and 103 bp, and three atypical introns of 210, 213 and 198 bp. The Euglena rpoC2 gene (830 codons) encodes a polypeptide with a predicted molecular weight of 94,628. The rpoC2 gene is interrupted by two group II introns of 580 and 514 bp, respectively. All of the exon-exon junctions were experimentally determined via cDNA cloning and sequencing analysis. Multiple protein alignments of the rpoC1 and rpoC2 gene products with related proteins from bacteria and chloroplasts were used to identify conserved regions. Transcripts from the rpoC1 and rpoC2 loci were characterized via Northern analysis. The rpoB, rpoC1 and rpoC2 genes are cotranscribed. Fully spliced tri-, di- and monocistronic transcripts were detected with hybridization probes specific for each gene. The relative abundance of the rpoC1 and rpoC2 transcripts is similar in RNA from dark- and light-grown Euglena. The mature 5'-ends of the rpoC1 and rpoC2 genes were mapped by primer extension. The 3'-end of the mature rpoC2 transcript was localized via an S1 nuclease protection assay. The rpoC1 and rpoC2 gene products were also compared to the largest subunits of RNA polymerases from archaebacteria and eukaryotes. The evolution of the Euglena genes is discussed.

PRDM16 is a transcriptional co-regulator that is highly expressed in HSCs and required for their maintenance. It is also involved in translocations in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) and T-cell acute lymphoblastic leukemia. Prdm16 is expressed as both full-length (f Prdm16) and short-length (s-Prdm16) isoforms, the latter lacking an N-terminal PR domain homologous to SET methyltransferase domains. The roles of both isoforms in normal and malignant hematopoiesis are unclear. In chromosomal rearrangements involving PRDM16, the PR domain is deleted. Furthermore, overexpression of s-Prdm16, but not f-Prdm16, can cause leukemia in a p53-/- background predisposed to malignancy. Based on this, s-Prdm16 has been proposed as an oncogene whereas f-Prdm16 has been suggested to possess tumor suppressor activity. The aim of this thesis was to more clearly elucidate the role of each Prdm16 isoform in normal and malignant hematopoiesis. We first showed that Prdm16 is essential for adult HSC maintenance using a conditional deletion mouse model specific for hematopoietic cells, as previous findings using an embryonic-lethal global Prdm16-/- mouse demonstrated this only in fetal liver. We then found, using a specific f-Prdm16-/- mouse model, that full-length Prdm16 is essential for HSC maintenance and induces multiple genes involved in GTPase signaling and represses inflammation. Based on a comparison of Prdm16-/- HSCs lacking both isoforms, and f-Prdm16-/- HSCs which express s-Prdm16, we were able to infer some hematopoietic properties of s-Prdm16 – namely that this isoform induces inflammatory gene expression and supports development of a Lineage-Sca1+cKit- lymphoid progenitor distinct from CLPs which predominantly differentiates into marginal zone B cells. s-Prdm16 expression alone, however, was not sufficient to maintain HSCs. We used a mouse model of human MLL-AF9 leukemia and found that leukemia derived from Prdm16-deficient HSCs had extended latency, although expression of Prdm16 decreases during MLL-AF9 transformation and is undetectable in ex vivo leukemic cells. Forced expression of f-Prdm16 in these cells further extended leukemic latency, while forced expression of s-Prdm16 shortened latency. Gene expression profiling using RNAseq indicated that forced expression of f-Prdm16 resulted in altered respiratory metabolism of MLL-AF9 cells, whereas expression of s-Prdm16 induced a strong inflammatory gene signature, comparable to that seen in HSCs expressing only s-Prdm16. Several inflammatory cytokines and chemokines induced by s-Prdm16 are associated with MDS and with a worse prognosis in human AML. Furthermore, leukemia expressing s-Prdm16 had an elevated number of cells with abnormal nuclei, characteristic of dysplasia. Finally, we performed an analysis of PRDM16 in human AML from the publically-available Cancer Genome Atlas dataset, containing clinical and gene expression data for 179 cases of AML. PRDM16 expression negatively correlated with overall survival, both in the entire dataset and in the NPM1 mutated and MLL¬-rearranged subsets, and s-PRDM16 exhibited a stronger correlation than f-PRDM16. HOX gene expression correlated with PRDM16 expression, suggesting that HOX genes may positively regulate PRDM16 expression in AML. In NPM1-mutant and MLL-rearranged subsets of AML, we also found that high PRDM16 expression correlated with an inflammatory gene signature, thus corroborating our findings in mouse MLL-AF9. Our findings demonstrate distinct roles for Prdm16 isoforms in both normal hematopoiesis and AML, and identify s-Prdm16 as one of the drivers of prognostically-adverse inflammatory gene expression in leukemia.

Xing, Lei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 111-120). / Abstracts in English and Chinese. / ABSTRACT OF THESIS ENTITLED: --- p.I / 論文摘要 --- p.Ill / ACKNOWLEDGEMENTS --- p.IV / ABBREVIATIONS --- p.V / CONTENTS --- p.VI / LIST OF FIGURES --- p.X / LIST OF TABLES --- p.XII / Chapter CHAPTER 1: --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.1.1 --- Pectic substances --- p.1 / Chapter 1.1.2 --- Structure and classification of pectins --- p.2 / Chapter 1.1.3 --- Classification of pectinases --- p.4 / Chapter 1.1.4 --- Application of pectinases --- p.5 / Chapter 1.1.5 --- Production of pectinases --- p.5 / Chapter 1.2 --- Lentinula edodes as a source of pectinolytic enzymes --- p.12 / Chapter 1.2.1 --- Taxonomy and Life cycle of L. edodes --- p.12 / Chapter 1.2.2 --- Pectin-degrading enzymes in L edodes --- p.13 / Chapter 1.3 --- Expression systems for fungal pectinolytic enzymes --- p.16 / Chapter 1.4 --- Gene expression analysis --- p.19 / Chapter 1.5 --- Objectives and Long-term significance --- p.20 / Chapter CHAPTER 2: P --- EGTINASES IN L. EDODES --- p.23 / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.2 --- Materials and Methods --- p.25 / Chapter 2.2.1 --- Fungal strains and growth conditions --- p.25 / Chapter 2.3.2 --- Gene models --- p.25 / Chapter 2.2.4 --- Enzyme activity assays --- p.26 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Alignment of 24 candidate pectin-degradation gene models --- p.30 / Chapter 2.3.2 --- Conserved domains in protein sequences of 24 gene models --- p.30 / Chapter 2.3.3 --- Signal Peptide prediction of pectin-degradation gene models --- p.30 / Chapter 2.3.4 --- Pectinases activities in L. edodes --- p.31 / Chapter 2.3.5 --- Growth of mycelia of L. edodes on pectin and non-pectin media --- p.31 / Chapter 2.4 --- Discussion --- p.48 / Chapter 2.4.1 --- Elimination of non-pectinolytic genes --- p.48 / Chapter 2.4.2 --- Conserved domains and active sites of 6 polygalacturonases --- p.49 / Chapter 2.4.3 --- Pectinases activities in L. edodes --- p.49 / Chapter 2.4.4 --- Effect of pectin on the growth of mycelia in L. edodes --- p.50 / Chapter 2.5 --- Conclusion --- p.51 / Chapter CHAPTER 3: --- TRANSCRIPTIONAL PROFILING OF PECTINASES GENES IN L. EDODES --- p.52 / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Materials and Methods --- p.57 / Chapter 3.2.2 --- Strain cultivation --- p.57 / Chapter 3.2.3 --- RNA extraction and first strand cDNA synthesis --- p.58 / Chapter 3.2.4 --- Quantitative RT-PCR --- p.58 / Chapter 3.2.5 --- Data analysis --- p.59 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.2 --- RNA quality of various samples in L. edodes --- p.62 / Chapter 3.3.3 --- Transcription of 14 putative pectinases genes --- p.62 / Chapter 3.3.4 --- Transcription profiling of pectinases genes during the development of L. edodes --- p.62 / Chapter 3.3.5 --- Transcriptional levels of pectinases genes in mycelia of L. edodes grown in different media --- p.63 / Chapter 3.4 --- Discussions --- p.73 / Chapter 3.4.2 --- Transcription profiling of pectinases genes in L. edodes during four developmental stages --- p.73 / Chapter 3.4.3 --- Differential transcriptional levels of pectinases genes in L. edodes mycelia grown in two media --- p.73 / Chapter 3.4.4 --- Effect of pectic substrates on the pectinases genes transcription in mycelia of L. edodes --- p.74 / Chapter 3.5 --- Conclusion --- p.76 / Chapter CHAPTER 4: --- CLONING OF PECTINASES GENES AND THEIR HETEROLOGOUS EXPRESSION IN PICHIA PASTORIS --- p.77 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Materials and methods --- p.79 / Chapter 4.2.2 --- Strain cultivation --- p.79 / Chapter 4.2.3 --- RNA extraction and first strand cDNA synthesis --- p.79 / Chapter 4.2.4 --- Cloning and sequencing of pectinases genes --- p.80 / Chapter 4.2.5 --- Subcloning and expression vector construction --- p.80 / Chapter 4.2.6 --- Growth of Pichia pastoris strains --- p.81 / Chapter 4.2.7 --- Transformation into P. pastoris and vivo screening of multiple inserts --- p.81 / Chapter 4.2.8 --- Expression of recombinant P. pastoris strains --- p.82 / Chapter 4.2.9 --- RNA extraction and transcription analysis of pectinases genes in recombinant Pichia strains --- p.83 / Chapter 4.2.10 --- Enzyme activity assays --- p.83 / Chapter 4.2.11 --- SDS-PAGE --- p.84 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.2 --- RT-PCR for full-length cDNA of 13 pectinases genes --- p.88 / Chapter 4.3.3 --- Cloning and sequences analysis of 4 putative pectinases genes --- p.88 / Chapter 4.3.4 --- Construction of expression vectors of pectinases genes and transformation to P. pastoris --- p.88 / Chapter 4.3.5 --- Screening of multiple inserts clones --- p.88 / Chapter 4.3.6 --- Recombination and integration of pectinases genes in P. pastoris --- p.89 / Chapter 4.3.7 --- Transcription and expression of pectinases genes in recombinant Pichia strains. --- p.89 / Chapter 4.4 --- Discussion --- p.104 / Chapter 4.4.2 --- cDNA sequences of 4 pectinases genes --- p.104 / Chapter 4.4.3 --- Heterologous expression of 2 pectinases genes in P. pastoris --- p.104 / Chapter 4.4.4 --- Characterization of the pectinases expressed by recombinant Pichia strains --- p.106 / Chapter 4.5 --- Conclusion --- p.108 / Chapter CHAPTER 5: --- CONCLUDING REMARKS --- p.109 / REFERENCES --- p.121

Phosphatase and tensin homologue deleted on chromosome ten (PTEN) is one of the most commonly altered tumor suppressors in human cancer. It is a dual-specificity phosphatase that by converting the lipid second messenger PIP3 to PIP2 antagonizes the PI3K/AKT signaling pathway. PTEN also has numerous, albeit controversial nuclear functions, which thus far have been shown to be independent of its phosphatase activity. Although a number of studies have described that loss or gain of PTEN protein expression alters gene expression patterns, relatively little is known about the exact mechanism. In this research study, we investigated PTEN’s influence on gene expression and its role in transcription regulation. First, we established mouse embryonic fibroblasts (MEFs) as a suitable model system to study the effects of PTEN loss on gene expression. Using an Adeno-virus containing Cre-recombinase, Pten expression could be ablated efficiently in MEFs carrying loxP sites flanking exon 5 of the endogenous Pten locus. Genome-wide mRNA microarray analysis revealed that Pten deletion decreased the transcript levels of a subset of genes and increased the transcript levels of a different subset of genes. Moreover, by uncoupling these effects from PTEN’s role in the PI3K/AKT pathway we discovered that Pten loss can alter gene expression in a PI3K/AKT-dependent as well as a PI3K/AKT-independent manner. The upregulated genes were enriched for genes involved in DNA binding, replication, and repair, but also for regulation of gene expression. Gene expression can be influenced by histone modifications. However, loss of PTEN did not affect histone modifications globally as evidenced by western blotting. Using native ChIP-Seq experiments we showed that loss of PTEN altered the levels of H3K36me3 and H3K27me3 on a subset of genes and markedly decreased levels of H3K27ac at most enhancers as well as super-enhancers. However, RNAPII occupancy on enhancer-associated genes did not decrease, suggesting that the modulation of enhancer strength did not affect RNAPII recruitment to TSS. In Chapter 3 we identify a nuclear pool of Pten that could associate with chromatin. Furthermore, we are the first to report that nuclear PTEN can directly interact with components of the transcription machinery including CDK7, CDK9, Cyclin T1, AFF4, and RNAPII. Loss of PTEN increased phosphorylation of Ser2 and Ser5 of the RNAPII CTD as well as RNAPII occupancy on promoters of expressed genes indicating an increase in transcriptional activity in PTEN-/- cells. Furthermore, PTEN deletion resulted in the upregulation of genes which are part of the important “Achilles cluster”, previously shown to confer sensitivity to transcription inhibition. We believe that it is over-expression of those genes that render PTEN deficient cells especially sensitive to transcription inhibitors such as THZ1, Triptolide, Flavopiridol and LDC000067. Over-expression of wild type PTEN but not a phosphatase-dead mutant of PTEN could decrease cells’ sensitivity to treatment with THZ1 or Flavopiridol. It also decreased protein levels of p-AKT Ser473 as well as RNAPII Ser2P and Ser5P suggesting that the phosphatase activity of PTEN is important for its role in transcription regulation. In sum, we propose a model in which PTEN binds to CDK7, CDK9, Cyclin T1, RNAPPII and/or AFF4 thereby exerting a negative regulatory effect on the activity of transcription complexes. Upon loss of PTEN the negative regulatory effect is eliminated and transcription of a subset of genes increases. It is most likely these genes that confer sensitivity to transcription inhibition on PTEN-/- cells. The better understanding of this oncogenic mechanism may reveal novel therapeutic opportunities, and ultimately we propose that the sensitivity of PTEN deficient cells to inhibitors of transcription could provide an effective clinical strategy to target PTEN deficient cancers.

RNA polymerase II (RNAP II) is responsible for transcribing messenger RNAs (mRNAs) as well as non-coding RNAs such as small nuclear RNAs (snRNAs) and microRNAs in eukaryotic cells. Rpb1, the largest catalytic subunit of this complex, possesses a unique C-Terminal Domain (CTD) that consists of tandem heptad repeats (the number varying from 26 to 52 by organism) with the consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). The CTD is extensively phosphorylated and dephosphorylated on non-proline residues during different steps of the transcription cycle, with roles for the threonine (Thr4) and tyrosine (Tyr1) attracting more attention. For example, in chicken cells, Thr4 functions in histone mRNA 3’ end formation, and Tyr1 phosphorylation is primarily associated with promoters and upstream antisense RNA formation, as well as preventing degradation of the polymerase, processes not found across all eukaryotes. A detailed introduction is described in Chapter 1. Taking advantage of the genetic tractability of yeast cells, we created a yeast (S. cerevisiae) strain with all CTD threonines substituted with valines (T4V) to study the role of CTD Thr4 in transcription in yeast, which prior to this study has been poorly characterized in S. cerevisiae. Using the T4V strain, we found that Thr4 was required for proper transcription of phosphate-regulated (PHO) and galactose-inducible (GAL) genes. We found genetic links between the T4V polymerase and genes encoding subunits of the Swr1 and Ino80 chromatin remodeling complexes, as well as the histone variant Htz1. We further provide evidence that CTD Thr4 is required for proper eviction of Htz1 by the Ino80 complex from genes requiring Thr4 for activation, presented in Chapter 2 of this thesis. Finally, Chapter 3 describes the functions of CTD Tyr1 in S. cerevisiae. Using a strategy similar to the T4V strain, I created a strain expressing an endogenous Rpb1 with all CTD tyrosine residues mutated to phenylalanine (Y1F). We found that this strain was viable, but with a severe slow-growth phenotype. We found genetic links between the Y1F polymerase and kinase/cyclin pair Srb10/Srb11, as well as an increase in occupancy on chromatin for the same. Further analysis indicated that RNA levels of genes associated with MAP Kinase associated stressors were dysregulated, and poly(A) site selection was biased towards distal poly(A) sites. Next, using an in vitro kinase assay, we showed Tyr1 phosphorylation on the CTD by MAP kinase Slt2, and in vivo CTD Tyr1 phosphorylation levels changed based on Slt2-associated stress response, as well as a decrease in in vivo Tyr1P-RNAP II from an Slt2 kinase-dead strain. Analysis of termination factors Nrd1 and Rtt103 showed transcription termination defects were likely the result of disruption of the interaction between the CTD interacting domains of these two proteins and the Y1F CTD. Extending this, we found additional disruptions in Slt2 recruitment to chromatin, increasing the depth of our knowledge of the interplay between induction of stress-associated genes, Slt2 function, and Nrd1-mediated termination.

Luo, Xiao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 118-134). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.iv / Abbreviations --- p.v / Contents --- p.vi / List of Figures --- p.ix / List of Tables --- p.xii / Chapter Chapter 1 : --- Literature Review --- p.1 / Chapter 1.1 --- Lentinula edodes --- p.1 / Chapter 1.1.1 --- Introduction and taxonomy --- p.1 / Chapter 1.1.2 --- Nutritional values and medical values --- p.2 / Chapter 1.2 --- Life cycle and morphology --- p.5 / Chapter 1.3 --- Lignocellulolytic system in wood-rotting fungi --- p.9 / Chapter 1.3.1 --- Structures of lignin --- p.9 / Chapter 1.3.2 --- Wood-rotting fungi --- p.11 / Chapter 1.3.3 --- Lignin degradation by white rot fungi --- p.12 / Chapter 1.3.4 --- Ligninolytic enzymes --- p.16 / Chapter 1.3.4.1 --- Lignin peroxidase --- p.16 / Chapter 1.3.4.2 --- Maganese peroxide --- p.16 / Chapter 1.3.4.3 --- Laccases --- p.19 / Chapter 1.3.5 --- Potential Industrial application of liglinolytic enzymes --- p.22 / Chapter 1.3.6 --- Ligninolytic enzymes in L. edodes --- p.23 / Chapter 1.4 --- Expression systems for fungal ligninolytic enzymes --- p.24 / Chapter 1.5 --- Aim of this project --- p.27 / Chapter 1.6 --- Long-term significance --- p.28 / Chapter Chapter 2: --- Sequence analysis of ligninolytic enzymes from Lentinula edodes --- p.29 / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and methods --- p.32 / Chapter 2.2.1 --- Phylogenetic study and signal peptide prediction of the decuced ligninolytic enzymes --- p.32 / Chapter 2.2.2 --- Comparison ligninolytic enzymes of L. edodes and other basidiomycetes fungi --- p.32 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- Protein sequence analysis and signature sequences identification of L. edodes laccases --- p.34 / Chapter 2.3.2 --- Protein sequence analysis of L. edodes manganese peroxidases --- p.34 / Chapter 2.3.3 --- Phylogenetic study of ligninolytic genes from L.edodes --- p.35 / Chapter 2.4 --- Disscussion --- p.52 / Chapter Chapter 3: --- Transcription profiling of ligninolytic enzymes from Lentinula edodes --- p.56 / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.61 / Chapter 3.2.1 --- Strain cultivation --- p.61 / Chapter 3.2.2 --- "RNA extraction, mRNA isolation and cDNA synthesis" --- p.63 / Chapter 3.2.3 --- RNA Quality Estimation --- p.64 / Chapter 3.2.4 --- cDNA synthesis --- p.65 / Chapter 3.2.5 --- Primer verification --- p.66 / Chapter 3.2.6 --- Quantitative RT-PCR --- p.66 / Chapter 3.3 --- Results --- p.70 / Chapter 3.3.1 --- RNA quality estimation --- p.70 / Chapter 3.3.2 --- Quantification real time PCR --- p.70 / Chapter 3.3.3 --- Transcriptional profiling of laccases during the development of L edodes --- p.70 / Chapter 3.3.4 --- Transcriptional profiling of MnPs during the development of L edodes --- p.71 / Chapter 3.3.5 --- Transcript level analysis of laccases from in mycelia grown on lignocelluloses medium and non -lignocelluloses medium --- p.71 / Chapter 3.3.6 --- Transcript level analysis of MnPs in mycelia grown on lignocelluloses medium and non -lignocelluloses medium --- p.72 / Chapter 3.3.7 --- Differential expression of laccases from L. edodes grownin lignocelluloses medium during mycelia stage --- p.72 / Chapter 3.3.8 --- Differential expression of laccases from L. edodes grownin lignocelluloses medium during mycelia stage --- p.72 / Chapter 3.4 --- Discussion --- p.87 / Chapter 3.4.1 --- Transcriptional profiling of laccases and MnPs during four developmental stages --- p.87 / Chapter 3.4.2 --- Transcriptional profiling of laccases and MnPs in mycelium grown in lignocelluloses and non-lignocelluloses medium --- p.88 / Chapter 3.4.3 --- Temporal differential expression of laccases and manganese peroxidases --- p.90 / Chapter 3.5 --- Conclusion --- p.92 / Chapter Chapter 4: --- "Cloning and heterologous expression of Lentinula edodes laccase, lac1B, in yeast Pichia pastoris" --- p.93 / Chapter 4.1 --- Introduction --- p.93 / Chapter 4.2 --- Materials and Methods --- p.95 / Chapter 4.2.1 --- Strain cultivation --- p.95 / Chapter 4.2.2 --- First strand cDNA synthesis --- p.95 / Chapter 4.2.3 --- Construction of cDNA library --- p.95 / Chapter 4.2.4 --- Signal peptide prediction of Iac1 B --- p.96 / Chapter 4.2.5 --- Cloning of native laccase into Pichia pastoris expression vector --- p.96 / Chapter 4.2.6 --- Screening for positive colonies --- p.97 / Chapter 4.2.7 --- Construction of pool of recombinant vector --- p.97 / Chapter 4.2.8 --- Transformation of P. pastoris --- p.98 / Chapter 4.2.9 --- Screening for expression cassette into Pichia pastoris --- p.98 / Chapter 4.2.10 --- Enzyme Activity assay --- p.99 / Chapter 4.2.11 --- SDS-PAGE --- p.100 / Chapter 4.3 --- Results --- p.103 / Chapter 4.3.1 --- Screening for positive colonies with recombinant vector in TOP10 --- p.103 / Chapter 4.3.2 --- Screening for expression cassette from transform ants of P pastoris --- p.103 / Chapter 4.3.3 --- Enzyme activity assay --- p.103 / Chapter 4.3.4 --- SDS-PAGE --- p.104 / Chapter 4.4 --- Disscussion --- p.109 / Chapter Chapter 5: --- Concluding Remarks --- p.111 / Reference --- p.118

Transcription, the first step in gene expression, is a highly regulated process which relies on a multi-protein complex to occur. Among these proteins are transcription factors, including initiation and elongation factors, which play differing roles in early and late stages of transcription. The mechanisms of transition from transcription initiation to elongation are not well understood in archaea, nor are the structures of the transcription factors involved. For transcription to occur in vitro, transcription factors TATA binding protein (TBP) and Transcription Factor B (TFB) are sufficient to allow RNA polymerase (RNAP) to synthesize RNA from template DNA. Another factor, Transcription Factor E (TFE), can also join the initiation complex and is likely to be essential in vivo. TFE is known to contribute to initiation by enhancing promoter opening, and while it has been shown to persist in elongation complexes, its role after initiation is unknown. Spt4/5, the archaeal homolog of the only universally conserved RNAP-associated factor, is known to join complexes in elongation steps and enhance processivity of the polymerase. However, if Spt4/5 joins pre-initiated complexes, it has been shown to inhibit transcription activity. The experiments in this thesis show that TFE and Spt4/5 participate in a crucial interchange at the upstream fork of the transcription bubble that helps define the timing of Spt4/5 binding. Using unnatural amino acid crosslinking techniques, the points of proximity between specific regions of these two factors and the template DNA have been mapped to identify possible sites of interaction. Competitive crosslinking assays indicate the exact timing of the shift in affinity between TFE and Spt4/5 for their shared binding site on RNAP. These data, combined with transcription assays, suggest a new role for TFE in preventing premature Spt4/5 binding, corresponding with a unique localized mobility within the winged helix of TFE.