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Title:Biochemical and structural studies of RNA damage and repair in bacteria
Author(s):Carruthers, Amy Lynn
Director of Research:Huang, Raven
Doctoral Committee Chair(s):Huang, Raven
Doctoral Committee Member(s):Gerlt, John; Gennis, Robert; Jin, Hong
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):RNA
ribotoxins
RNA ligase
nucleotidyltransferase
RNA repair
Abstract:In an environment with limited resources, organisms employ a variety of offensive and defensive mechanisms to ensure their own survival and growth. Many organisms utilize ribotoxins to respond to cellular stress or to kill competing organisms. Ribotoxins are site-specific endonucleases which typically target RNA molecules that are necessary for translation mainly, tRNAs and rRNAs which can result in their loss of function. In order to respond to RNA damage, some organisms have developed RNA repair systems which can heal and seal the cleaved RNA molecule to restore its function. For example, two proteins in bacteriophage T4, PnkP and Rnl, have been shown to reverse tRNA damage caused by its host’s stress response. In bacteria, numerous RNA repair systems have been identified by bioinformatics and biochemical studies; however, the in vivo targets of these systems have been difficult to identify. RNA repair systems repair systems are highly diverse but are generally composed of an RNA ligase and several processing and scaffold proteins. One family of RNA ligases, RtcB, is widely distributed among the domains of life and has been implicated in tRNA splicing in eukaryotes and archaea. However, the role of RtcB in bacteria remains elusive. Bacterial RtcB may be involved in RNA repair but the complexity of RNA damage and repair within the cell make it difficult to identify the in vivo targets of a particular RNA repair system. Instead, our lab has chosen to study proteins that are frequently found to be associated with RtcB in order to learn about RtcB itself. One such protein which has been found to be in many diverse RNA repair operons, including many containing RtcB, is RlaP-NTase (RNA Ligase-associated Polβ). RlaP-NTase is a novel nucleotidyltransferase (NTase) of the Polβ superfamily. NTases are known to transfer NMP from an (d)NTP onto an acceptor molecule, such as an RNA, DNA, protein, or small molecule. The Polβ superfamily is very large and highly diverse family which carries out many important biological functions including tRNA and mRNA maturation, DNA repair, cell signaling, and innate immune response. Due to its association with RtcB and its likely NMP transfer activity, we hypothesized that RlaP-NTase be involved in cellular response to “smart” ribotoxins which not only cleave RNA but also remove one or two nucleotides from the damaged ends. In order to study RlaP-NTase, we chose to clone and purify recombinant RlaP-NTase from two organisms, pseudomonas fluorescens and pseudomonas aeruginosa. Biochemical studies of these two enzymes have shown that RNAs, not DNAs or proteins, are substrates for RlaP-NTase. Additionally, in vitro assays employing two synthetic RNA molecules revealed that RlaP-NTase catalyzes the addition of one or two NMP molecules onto the 3’-OH group of its RNA substrate and that the reaction exhibits a preference for RNA substrates resulting from damage by ribotoxins. Interestingly, RlaP-NTase preferred to utilize Mn+2 as a metal cofactor for coordinating NTP whereas most known NTases use Mg+2. Structural studies of PfRlaP-NTase have also been carried out. The structure of PfRlaP-NTase was revealed to be an N-terminal NTase motif fused to a C-terminal helical domain. The N-terminal and C-terminal domains come together to form a cleft where NTP and RNA substrate bind. The NTase fold contains catalytic aspartic acid residues positioned to coordinate NTP. Furthermore, based on the results of our in vitro reconstitution assays, we carried out preliminary functional studies of PfRlaP-NTase. Aminoacylation assays indicated that RlaP-NTase is necessary to restore biological function to repaired tRNAs that have experienced loss of a single nucleotide. While this study was not conclusive, it is consistent with our hypothesis that RlaP-NTase acts to restore nucleotides removed by “smart” ribotoxins prior to RNA repair resulting in a functional RNA once ligated. In addition to studying RNA repair, our lab is interested in studying the purposeful damage of RNA carried out by ribotoxins. Though bioinformatics has identified many new ribotoxin families present in all major bacterial lineages, only a handful of ribotoxins have been thoroughly characterized. Previous work in our lab has involved cloning many of these uncharacterized ribotoxins and testing them for toxicity in vivo. One novel ribotoxin which was shown to be highly toxic was Pa-CD (pseudomonas aeruginosa colicin D-like). RNA-seq assays performed in our lab determined that Pa-CD cleaves four isoaccepting tRNAs between bases 38 and 39 in the anticodon loop. I have cloned and overexpressed Pa-CD in complex with its immunity protein, Pa-CD Immunity and separated the tightly bound proteins. I performed biochemical assays to confirm the target of Pa-CD in vitro and carried out structural studies of the purified complex.
Issue Date:2019-07-09
Type:Text
URI:http://hdl.handle.net/2142/105789
Rights Information:Copyright 2019 Amy Carruthers
Date Available in IDEALS:2019-11-26
Date Deposited:2019-08


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