Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: Patrick M. Gaffney, College of Earth, Ocean, & Environment. Includes bibliographical references.

DNA 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. / text

Not available / text

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

The study of many of glycosyltransferases is limited due to an inadequate access to sugar nucleotides. Preparation of sugar nucleotides through the use of nucleotidylyltransferases with broad substrate specificities is gaining significant interest and offers high yields and stereospecificity. Physiologically, the glucose 1-phosphate thymidylyltransferase catalyzes the condensation of ?-D-glucose 1-phosphate and deoxythymidine triphosphate to yield deoxythymidine diphospho glucose. Exploiting and targeting these enzymes also has the potential of yielding new therapeutics. Cps2L is a thymidylyltransferase isolated from Streptococcus pneumoniae, with broad substrate flexibility. The substrate specificity of Cps2L was evaluated with new sugar 1-phosphate analogues to gain further insight into substrate and inhibitor requirements. Several sugar 1-phosphate analogues including sugar 1C-phosphonates (and analogues thereof), 2-deoxy-2-fluorosugar 1-phosphates, and glucopyranose 1- boranophosphates have been used to probe the sugar 1-phosphate modification tolerance of Cps2L. In addition, NMR spectroscopy was used to determine the anomeric stereochemistry of 2-deoxy-2-fluorosugars nucleotide products. For those substrates that were accepted by Cps2L, steady-state kinetic parameters were determined. The enzyme is able to almost equally form Michaelis complexes with different sugar substrates, whereas the turnover values for obtaining the corresponding sugar nucleotide were different. The evaluation of the substrate tolerance of Cps2L, as well as the synthesis of ?-D-glucose-1C-thiophosphonate, a difluoro and a bisphosphono analogue of ?-D-glucose 1C-phosphonate will be described.

C. cinereus, any two of which are sufficient to promote B-regulated development following cell fusion. The isolation of the B42 locus is described along with the DNA sequence analysis that identified nine B mating type genes within a 27kb B42 -specific DNA sequence. Six of the genes, with small transcripts of 800-900nt, encode the mating pheromone precursors and the other three, with 1.9 to 2-5kb transcripts, encode the transmembrane pheromone receptors. The genes are arranged in three groups, designated group 1, 2 and 3, each consisting of one receptor gene and two pheromone genes. B42 and B6 share the same alleles of the group 1 genes, but not those of groups 2 and 3. This was demonstrated by DNAsequence analysis and Southern blot analysis. None of the group 1 genes from B42 were able to activate B -regulated development in a B6 host when introduced by transformation but with one exception, all genes from group 2 and group 3 were able to do so. This analysis led to the recognition that the three genes in any one group are held together in an allele-specific DNA sequence and that Southern blot analysis and transformation can be used to identify shared alleles in uncloned loci. Extensive Southern analyses using cloned genes to probe genomic DNAs from strains having other B mating specificites showed that different B loci may share identical alleles of two groups of genes. Mating partners thus require different alleles of only one group of genes to generate a compatible B mating interaction. Transformation analyses with the same cloned genes confirmed the conclusions derived from the hybridisation data. Multiple B mating specificities thus appear to be derived from three groups of multiallelic and functionally redundant genes. A tenth gene located within the B42- specific DNA sequence encodes a putative transporter protein belonging to the major facilitator superfamily (MFS). In other genomic backgrounds this gene lies in homologous flanking sequences and its presence within the B42 locus is unlikely to be related to mating type function.

GTPases are found in all domains of life and are highly conserved. In eukaryotes, they serve as signalling molecules for many cellular processes. However, the prokaryotic GTPases play a very different role and are found to be associated with ribosome function. Among the 11 conserved GTPases, Der is the most interesting in prokaryotes. It possesses a unique structure with two GTPase domains (G-Domains) tethered by a variable length acidic linker and a carboxyl terminal KH-like domain. The exact function of Der is still under investigation and most of the data suggest that it is important for 50S ribosomal assembly or stability. In order to investigate the function of Escherichia coli Der (Ec-Der), expression plasmids for wild-type and mutated proteins were created and the proteins were successfully expressed. The expression of the mutant protein that lacked G-Domain 1 was toxic to the cells and it was found that some large ribosomal proteins were missing from the ribosomes of these cells. In addition, other macromolecular complexes such as the GroEL/GroES chaperonin appeared not to be assembled under these conditions. The activities of both wild-type and mutated proteins were also tested and found to be dependent on potassium ions (K+), which enhanced nucleotide binding. Additionally, intra-molecular control over nucleotide binding and release was also observed for Ec-Der. The in vitro selection of RNA aptamers with nanomolar affinity for Ec-Der produced aptamers that contained short variable sequences. These aptamers affected the growth of the E. coli cells and caused a change in cellular morphology that had been noted previously during Ec-Der over-expression. Ec-Der showed high affinity (nM) to both selected RNA and the unselected RNA library. The activity of Ec-Der and Era was inhibited in the presence of any sequence of RNA that has the length of greater than 16 nucleotides. RNA was also cross-linked to Ec-Der in the presence of GTP, but not GDP, suggesting that RNA was a regulator of the Ec-Der GTPase cycle. Based on these results, it is speculated that Ec-Der might be involved in more than one function. It may be acting at the level of the membrane (based on cellular morphology reported here and by Hwang and Inouye 2001) and may also take part in processes related to ribosome function. Regulation of protein activity by RNA length has not been predicted or described and this may represent a novel mean of regulation of the Era subfamily of GTPases.

Typescript (photocopy) / xii, 107 leaves, [11] 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.)--Dept. of Agricultural Science, University of Adelaide, 1982

Thesis (Ph.D.) - Department of Agricultural Science, University of Adelaide, 1982. / Typescript (photocopy).