Browsing by Subject "CTD"
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Item Arabidopsis Thaliana CARBOXYL-TERMINAL DOMAIN PHOSPHATASE-Like1 (CPL1) Mediates Responses to Iron Deficiency and Cadmium Toxicity(2014-04-24) Aksoy, EmreThe expression of genes that control iron (Fe) uptake and distribution (i.e., Fe utilization- related genes) is under a strict regulation. Fe deficiency strongly induces Fe utilization- related gene expression; however, little is known about the mechanisms that regulate this response in plants. In this dissertation, a RNA metabolism factor, RNA POLYMERASE II CTD-PHOSPHATASE-LIKE1 (CPL1) was shown to localize to the root stele, and to be involved in the regulation of Fe deficiency responses in Arabidopsis thaliana. An analysis of multiple cpl1 alleles established that cpl1 mutations enhanced transcriptional responses of Fe utilization-related genes, e.g. IRON-REGULATED TRANSPORTER1 (IRT1), to low Fe availability. In addition to the lower Fe content in the roots, but higher Fe content in the shoots of cpl1-2 plants, the root growth of cpl1-2 showed improved tolerance to Fe deficiency. Genetic data indicated that cpl1-2 likely activates Fe deficiency responses upstream of both FE?DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT)- dependent and -independent signaling pathways. Interestingly, various osmotic stress/ABA-inducible genes were up-regulated in cpl1-2, and the expression of some ABA-inducible genes was controlled by Fe availability. Unlike Fe, accumulation of the heavy-metal cadmium (Cd) in plants is toxic and it is absorbed by the roots due to the low selectivity of metal transporters such as AtIRT1. In this dissertation, CPL1 was also shown to regulate the transcriptional responses to Cd toxicity. cpl1-2 showed higher tolerance to the Cd toxicity by enhancing the root-to-shoot translocation of Cd by an unknown mechanism. A knowledge-based screening resulted in identification of a putative metal transporter, OLIGOPEPTIDE TRANSPORTER (OPT), which was highly induced in cpl1-2 upon exposure to Cd. OPT was localized to the plastids, indicating a role of plastids in Cd transport and accumulation. The root growth of opt mutants showed higher tolerance to the Cd toxicity, and the mutants accumulated less Cd, Fe and Zn, indicating the involvement of OPT in the transport of these metals. This presented dissertation suggests that 1) CPL1 functions as a negative regulator of the Fe deficiency signaling at the crosstalk with a branch of the osmotic stress/ABA signaling pathway, and 2) CPL1 regulates the Cd distribution in plants by repressing the expression of OPT.Item Phosphatases and prolyl-isomerase in the regulation of the C-terminal domain of eukaryotic RNA polymerase II(2012-12) Zhang, Mengmeng; Zhang, Yan Jessie; Robertus, Jon D.; Appling, Dean R.; Siegel, Dionicio R.; Fast, Walter L.In eukaryotes, the first step of interpreting the genetic information is the transcription of DNA into RNA. For protein-coding genes, such transcription is carried out by RNA polymerase II. A special domain of RNA polymerase II, called the C-terminal domain (CTD), functions as a master controller for the transcription process by providing a platform to recruit regulatory proteins to nascent mRNA (Chapter 1-2). The modifications and conformational states of the CTD, termed the 'CTD code', represent a critical regulatory checkpoint for transcription. The CTD, found only in eukaryotes, consists of 26--52 tandem heptapeptide repeats with the consensus sequence, Tyr₁Ser₂Pro₃Thr₄Ser₅Pro₆Ser₇. Phosphorylation of the serines and prolyl isomerization of the prolines represent two major regulatory mechanisms of the CTD. Interestingly, the phosphorylation sites are typically close to prolines, thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases. Understanding how those modifying enzymes recognize and regulate the CTD is important for expanding our knowledge on the transcription regulation and deciphering the 'CTD code'. During my PhD study, I studied the function of CTD phosphatases and prolyl isomerase in the CTD regulation using Scp1, Ssu72 and Pin1 as model regulators. Scp1 and Ssu72 are both Ser5 phosphatases. However, Ssu72 is an essential protein and regulates the global transcription while Scp1 epigenetically silences the expression of specific neuronal genes. Pin1 is a highly conserved phosphorylation-specific prolyl isomerase that recognizes the phospho-Ser/Thr-Pro motif within the CTD as one of its primary substrates in vivo. Among these enzymes, Scp1 is the focal point of this dissertation, as it was studied from different angles, such as enzymatic mechanism (Chapter 3 describes the capture of phospho-aspartyl intermediate of Scp1 as a direct evidence for the proposed two-step mechanism), specific inhibition (Chapter 4 describes the identification and characterization of the first specific inhibitor of Scp1), and its non-active-site contact with the CTD (Chapter 5 describes the structural basis of this contact). These studies are of great importance towards understanding the molecular mechanism of the dephosphorylation process of the CTD by Scp1.