description- – DNA repair is essential for survival, as damage to the genome can interrupt the precarious balance of cell functions, causing further mutations and possibly leading to cancer. The bacterial transcription repair coupling factor, Mfd, is capable of recognizing a stalled RNA polymerase at a site of DNA damage. The Mfd works both to remove the RNA polymerase through its motor function (utilizing the energy of ATP to translocate along DNA), and to recruit the DNA repair complex UvrA/B/C. To study conformational changes in the protein, we are creating multiple mutants of the full length Mfd protein. My approach is to use a cleavable mutant of full-length Mfd as a template for further mutations. This will allow us to probe for conformational changes by changing interactions at the interface of the two halves of Mfd, and then using the ability to cut with TEV protease as a sensor to identify and characterize the open state of the protein. By introducing this TEV protease cut site at residue 450 in the protein linker region between the N (amino-) and C (carboxy-) terminal domains, we can then assess the conformational changes Mfd must undergo to obtain activity. We can study the effect of further mutations on the full length and cut versions of the protein. Another approach attempted in this study involves using cysteine modification of the full length Mfd protein as a sensor for these conformational changes. Mfd acts as a model system for studying the DNA repair mechanisms found in humans, and the elucidation of functional and conformational changes in Mfd contributes to studying disease phenotypes resulting from aberrant transcription coupled repair.
subjectcollectiondatepublishercreatorformat description- – Cysteine dioxygenase (CDO) is a non-heme iron enzyme that can be found in mammalian tissue. It is mainly localized in the liver but is also present in the brain, kidney, and adipose tissue. CDO converts cysteine to cysteine sulfinic acid, which is the first step in cysteine metabolism in the human body. CDO contains a novel cofactor located near the metal binding site that is present in another enzyme, galactose oxidase, where it is essential for redox function. This suggests that the linkage may play an important role in CDO as well. The cofactor consists of Y157 and C93. Mutation of the C93S causes a drop in activity to 57.1% and a mutation of the Y157F causes a drop to 8.1%. The metal center was studied using XAS revealing that the addition of cysteamine, an activator of CDO, changes the conformation of the binding site significantly. CDO differs from the rest of the cupin super family in that it does not contain a 2-his-1-carboxylate binding motif but rather the carboxylate is replaced with another histidine. A mutation of one of the binding residues, H140D, caused the enzyme to be non-active. Also the mechanism of the CDO was studied by conducting activity assays with various inhibitors and activators that yielded contradicting results with previously published work.
subjectcollectiondatepublishercreatorformat description- – There is strong crystallographic evidence that the 23S rRNA is the only catalytic entity in the peptidyl transferase center. Various mechanisms for the catalysis of peptidyl transfer have been proposed. Recently, attention has been given to the idea that the 23S rRNA simply acts to position the tRNA for spontaneous peptidyl transfer and that chemical catalysis may play only a secondary role. Conserved nucleotides U2585 and U2506 are thought to be involved in positioning the 3' ends of A- and P-site substrates based on the crystallographic evidence, and because mutagenesis at these sites severely impairs peptide bond formation. In this study, pure populations of ribosomes with either U2585A or U2506G mutations in the 23S rRNA were analyzed to test the hypothesis that substitutions at nucleotides U2585 and U2506 in the peptidyl transferase center impair peptide bond formation by altering the position of the 3' end of P-site tRNA relative to the 23S rRNA. Pure populations of mutant or wild-type ribosomes were obtained by an affinity tagging system and probed with 32P-labeled [2N3A76]tRNAPhe to determine how the 3' end of tRNA interacts with the ribosomal proteins and 23S RNA at the peptidyl transferase center. Some of the data for the ribosomes with a G at position 2506 are consistent with a model suggested by Schmeing and coworkers in which nucleotide U2506 breaks from its original wobble base pair with nucleotide G2583 during A-site tRNA binding and swings towards the 3' end of P-site tRNA, while nucleotide U2585 simultaneously moves away from the 3' end of P-site tRNA.
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