Browsing by Subject "Nucleotides"
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Item Developmental responses of cotton genotypes to varying water application regimes(Texas Tech University, 2004-05) West-Emerson, Cora LeaNot availableItem Directed evolution of T7 RNA polymerase variants using an 'autogene'(2004-08) Chelliserrykattil, Jijumon Pavithran, 1974-; Ellington, Andrew D.Item Fidelity of nucleotide incorporation by the human mitochondrial DNA polymerase(2005) Lee, Harold Ray; Johnson, Kenneth AllenThe human mitochondrial DNA polymerase (pol γ) is a nuclearly encoded enzyme, imported to the mitochondria, solely responsible for the replication of the mitochondrial genome. I have characterized the kinetics of nucleotide incorporation, determined the discrimination constants for misincorporation, and calculated the overall fidelity of this enzyme. Additionally, I have investigated the dependence of these parameters on the concentration of magnesium ion present in the reaction. There have been reports in the literature of pol γ having reverse transcriptase activity, and of attempts to determine a physiological role for this activity. Indeed, many steady-state kinetic assays of this enzyme reported in the literature are reverse transcriptase assays. I have characterized the kinetics of incorporation of the reverse transcriptase activity of polymerase γ, both in single turnover and processive polymerization assays. Additionally, I have characterized the activity of the 3′-5′ exonuclease domain on a DNA/RNA heteroduplex. For many years there has been research into the factors that contribute to polymerase discrimination. Base pair hydrogen bonding, base stacking, steric interactions, active site tightness, and factors unknown are all believed to play a role. However, recent attempts to investigate the contribution to discrimination afforded by base pair hydrogen bonding in Klenow fragment, using “shape mimic” nucleoside analogs have led to the supposition that base pairing plays little, if any, role in discrimination. In order to investigate the contribution of base pair hydrogen bonding to discrimination in pol γ, I have characterized the incorporation of natural nucleotides opposite the dT analog 2, 4-difluorotoluene deoxynucleoside (dF) and the dA analog 9- (1-aza-4-methyl-benzimidazolyl)-1′-β-2′-deoxyriboside (dQ). Additionally, the kinetic parameters of incorporation of dF opposite dT, and of dQ and 4-methylbenzimidazole (dZ) opposite dA have been determined. The rates of 3′-5′ exonuclease removal of natural nucleosides paired opposite dF and dQ have been determined as well. The mitochondrial polymerase is the sole enzyme responsible for replication of the mitochondrial genome. In the absence of accurate and efficient replication by polymerase γ, several clinical pathologies are observed including cardiac and neural myopathy, mitochondrial myopathy, anemia, and potentially fatal lactic acidosis. These symptoms are known effects of oxidative damage to the mitochondrial genome and of toxicity associated with the treatment of HIV infection with nucleotide reverse transcriptase inhibitors. Additionally, several diseases have been associated with mitochondrial genome mutation and depletion including Parkinson’s disease and Alzheimer’s disease. A full understanding of the fidelity of the mitochondrial DNA polymerase and the mechanisms by which this fidelity is insured will aid in the understanding of diseases associated with mitochondrial damage and in the design of drugs used to fight HIV, lacking the potentially fatal mitochondria based toxicities.Item The molecular basis of nucleotide recognition for T7 DNA polymerase(2008-08) Jin, Zhinan, 1972-; Johnson, Kenneth AllenDNA replication demands extraordinary specificity and efficiency of catalysis from a DNA polymerase. Previous studies on several DNA polymerases suggested that a rate-limiting conformational change preceding chemistry accounts for the high specificity following the induced fit mechanism. However, the identity of this rate-limiting conformational change and how it contributes to the fidelity is still under debate. An important study of T7 DNA polymerase performed by Tsai and Johnson using a conformationally sensitive fluorophore (CSF) characterized a conformational change directly and presented a new paradigm for nucleotide selectivity. This thesis describes work to further characterize the underlying molecular basis regulating the conformational change by a combination of site-directed mutagenesis, transient kinetics and crystallography. One flexible segment (gly-ala-gly) within the fingers domain was mutated to (ala-alaala). The kinetic analysis on this mutant showed that the mutations decreased the forward rate of the conformational change reported by the fluorophore about 1200-fold but there was no significant change on the reverse rate. The data suggested that the movement of the fingers domain is not a rigid body motion but may be complex due to the movements of various helices within the fingers domain. Quantification of the kinetics of incorporation of correct and incorrect base pairs showed the decrease of fidelity mainly was from the decreased forward rate during correct nucleotide incorporation. The roles of three active site residues, K522, H506, and R518, which form polar interactions with [alpha]-,[beat]- and [gamma]-phosphates of the incoming nucleotide respectively, in conformational change and catalysis were also characterized. All the mutants showed a slower conformational change than the wild type enzyme. After this conformational change, there was a rate limiting step with a rate comparable to kpol measured by quench-flow experiments. Correct nucleotide binding caused an increase in fluorescence, suggesting that the conformational change of the fingers domain delivers incoming nucleotide to a misaligned status even for a correct nucleotide with each of the mutants. The data suggested that active site residues play important roles in maintaining a fast conformational change and an accurate alignment of the active site during correct nucleotide incorporation. Yellow crystals of CSF-labeled T7 DNA polymerase with DNA and correct nucleotide (closed complex), incorrect nucleotide (misaligned complex) or no nucleotide (open complex) were grown to good size and diffracted to 3 Å during X-ray data collection. The structures of these complexes are still under refinement.Item Reduction of nucleic acid in single cell protein by an endogenous polynucleotide phosphorylase(Texas Tech University, 1976-12) Yang, Huei-HsiungNot availableItem Rhodopsin kinase structure: different nucleotide-binding states and implications for mechanism of activation of a G protein coupled receptor kinase(2007-12) Singh, Puja, 1979-; Tesmer, John; Hackert, Marvin L.G protein coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins and downregulation. The desensitization of GPCRs is critical to render cells responsive to further stimuli and if not regulated can result in many pathophysiological processes including heart abnormalities and hypertension. How GRKs recognize and are activated by GPCRs are not known, in part because the critical N-terminus and the kinase C-terminal extension were not resolved in GRK2 and GRK6 structures. The long-term goal of this project was to address this question by structural analysis of rhodopsin kinase (also known as GRK1), which represents a model system for studying phosphorylation-dependent desensitization of activated GPCRs. Herein we report structures of GRK1 from six crystal forms that represent three distinct nucleotide-ligand binding states. One of the (Mg²⁺)₂·ADP·GRK1 structures is the most high-resolution structure (1.85 Å) of a GRK to date. In one (Mg²⁺)₂·ATP·GRK1 structure, almost the entire N-terminal region (residues 5-30) is observed. In addition, different segments of the kinase C-terminal extension are ordered in the various nucleotide-bound structures. Together, these two elements form a putative receptor-docking site adjacent to the hinge of the kinase domain. Based on these structures, a model is proposed for how GRK1 interacts with activated rhodopsin and how rhodopsin binding in turn could activate the kinase. Two novel phosphorylation sites were also identified at the N-terminus. The physiological role of phosphorylation sites and the extensive dimerization interface mediated by the regulator of G protein signaling (RGS) homology domain of GRK1 was assessed using site-directed mutagenesis. In addition to mediating interaction with activated GPCRs, the N-terminus of GRKs also forms a binding site for calcium sensing proteins. Although its physiological significance is debated, the structures of these complexes could lend further insights into the conformation of the N-terminus of GRKs. The second chapter deals with attempts to isolate Ca²⁺·recoverin·GRK1 and Ca²⁺·calmodulin·GRK6 complexes. Finally, the RH domain of GRK2 binds G[alpha subscript q], G[alpha]₁₁, and G[alpha]₁₄ subunits thereby blocking their interactions with the downstream effectors. The third chapter involves attempts to isolate a complex of GRK6 and G[alpha]₁₆, a member of G[alpha subscript q] family.Item Structure-function studies with the cAMP receptor protein of Escherichia coli(Texas Tech University, 2003-05) Tutar, YusufCyclic AMP Receptor protein (CRP) regulates the transcription of more than 100 genes. In the absence of cAMP, CRP is inactive. Cyclic AMP binding induces a structure change in CRP that promotes its interaction with RNA polymerase and DNA. CRP is a dimer of identical subunits; each consisting of 209 amino acids. A CRP subunit is composed of two domains. The larger N-terminal domain binds the allosteric effector, cAMP. This domain consists of eight j8-sheets that provide a hydrophobic pocket for cAMP binding. One cAMP is bound to each subunit contacting amino acid residues from both subunits. The C-terminal domain contains a helix-tum-helix motif that binds specific DNA sequences. The structure of CRP in the absence of cAMP is unknown, therefore the details of the allosteric mechanism mediated by cAMP remain obscure. The allosteric conformational change in CRP upon binding cAMP can be understood by comparing CRP and the CRP-cAMP complex by similar biophysical characterization. Several groups have used Raman spectroscopy, and circular dichroism techniques to compare these two different states of CRP. These methods used high concentrations of salt to improve CRP solubility. Our laboratory has used Fourier Transform Infrared Spectroscopy (FTIR) along with STIR cards to overcome the problems of protein solubility and high salt concentration. Analysis of the Amide I region indicated a secondary structure distribution of 35% a-helix, 31% jS-sheet, 21% turn, and 13% unordered for both states of WT and its E72D, E72Q, and R82Q mutants. This result is consistent with X-ray analysis of CRP-CAMP2 (37% a-helix, 36% ^-sheet). Fluorimetric binding studies showed that cAMP binding exhibits negative cooperativity in cAMP binding to the second subunit and amino acid substitution at positions 72 and 82 reduced binding affinities for cAMP by factors of 2 to 25 fold. DNA binding studies indicated that the equilibrium constants of the mutant CRP: cAMP complexes measured for lac? were reduced compared to that of WT CRP: cAMP complex. In addition, the mutant complexes failed to footprint in the presence of RNA polymerase. The level of j8-galactosidase expression in the mutants varied depending on this negative allostery. Since, under the conditions utilizied in this study, cAMP makes no contact with the DNA-binding domains, it cannot induce a conformational change in them by direct interaction. This suggests that cAMP induces a change in the relative orientation of the two subunits because it binds close to the subunit interaction area. This change could be relayed to the DNA binding domain and could change the relative position and orientation of the recognition helices and the activity. Thus these results can explain the allosteric transition mediated by the binding of cyclic AMP that converts CRP from a protein having low DNA activity to one that exhibits high, sequence-specific, affinity for DNA