Characterization of RNA damage and repair in bacteria

Raybarman, Adrika

Permalink

https://hdl.handle.net/2142/124642 Copy

Description

Title

Characterization of RNA damage and repair in bacteria

Author(s)

Raybarman, Adrika

Issue Date

2024-04-17

Director of Research (if dissertation) or Advisor (if thesis)

Huang, Raven

Doctoral Committee Chair(s)

Huang, Raven

Committee Member(s)

• Cronan, John
• Fratti, Rutilio
• Zhang, Yan
• Zhao, Huimin

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• RNA damage
• RNA repair
• Bacteria
• RtcB
• tRNA splicing

Abstract

Competition among species for limited resources is a fundamental biological phenomenon. Microbes utilize a wide range of toxins to gain an advantage in the race for survival. A class of protein toxins called ribotoxins, which carry out site-specific cleavage of some essential RNAs, aid microorganisms in this endeavor. Ribotoxins can target RNAs involved in protein translation; for example, Colicin E3 targets the 16S rRNA, while Colicin D and Colicin E5 cleave tRNAs. The attacked species, in turn, have evolved RNA repair systems to counteract the effects of the damage. This thesis aims to understand better how the interplay of RNA damage and repair occurs. RtcB, which can ligate a 2’,3’cyclic phosphate and 5’OH, usually generated by ribotoxins, has been demonstrated to be involved in tRNA splicing in both Archaea and eukaryotic organisms. A sequence similarity network (SSN) showed that the majority of the RtcB proteins are present in bacterial genomes. Since bacteria do not have introns (a few exceptions include species with self-splicing introns in tRNA genes), we hypothesized that bacterial RtcBs may play a role in RNA repair to counteract the damage inflicted by ribotoxins. Therefore, the first part focuses on elucidating the biological role of RtcB proteins in bacteria. Two candidates from E. coli, hereafter named EcoRtcB1 and EcoRtcB2, belonging to two different clusters, were chosen for the initial study. To determine substrate specificity, in vitro ligation reactions were carried out using EcoRtcB1 and EcoRtcB2 for three types of RNA substrates: tRNAAsp cleaved at position 34 by Colicin E5, tRNAArg cleaved at position 38 by Colicin D, and 30S ribosomal subunit damaged in the decoding center by CdiA-CTECL. The results showed that EcoRtcB1 efficiently repaired the two tRNA substrates but did not repair the cleaved 16S rRNA substrate. On the other hand, EcoRtcB2 could efficiently ligate the damaged 30S substrate; however, it did not show any repair for either of the two tRNA substrates. These in vitro reaction results imply that the biological roles of the two proteins may be distinct. Moreover, EcoRtcB2 is present in a two-component operon encoding RtcB and PrfH (a predicted ribosome-rescuing factor). Additional in vitro and in vivo experiments with EcoRtcB2 and EcoPrfH demonstrated that repair of the 30S subunit in the context of the 70S ribosome was enhanced in the presence of PrfH, indicating that PrfH aids in dissociation. Further bioinformatic analysis showed that in three of the clusters generated by the SSN, the two-component operon encoding a distant RtcB and PrfH is present in bacteria. Another such candidate, CgiRtcB (C. gingivalis RtcB), was chosen from a previously studied organism in our lab, which is present in a different cluster from that of EcoRtcB2. We tested the 30S ribosomal subunit repair capabilities of CgiRtcB and observed efficient repair. However, there was a difference. The first step of the repair reaction by RtcB is a nucleotidyl transfer step where RtcB reacts with GTP to form a covalent RtcB-histidine-GMP intermediate. All E. coli RtcBs and other RtcBs described so far are GTP-dependent. So, we were surprised to see that CgiRtcB is ATP-dependent. Candidates were chosen from each of the eight clusters (based on the SSN), and reactions with alpha-P32-ATP and alpha-P32-GTP showed that members from three clusters were ATP-dependent. Further analysis showed that the C-terminal sequence of RtcBs may indicate ATP or GTP dependence. Efforts are currently underway to obtain the crystal structure of an ATP-dependent RtcB to understand how AMP interacts with the active site residues. The damaged RNA substrates studied so far focused on tRNA and 16S rRNA cleavage. However, there is another popular target for ribotoxins in the larger ribosomal subunit. Even though alpha-sarcin, a fungal ribotoxin produced by the Aspergillus giganteus mold, targets the eukaryotic 28S rRNA by cleaving one phosphodiester bond in the conserved sarcin-ricin loop (SRL), it is not efficient in cleaving the bacterial 23S rRNA. Therefore, we employed a strategy of screening alpha-sarcin homologs to search for more effective sarcin-like toxins for bacteria. These potential toxins were tested for toxicity. One of the candidates, sarcin-like 2 (SL-2), from the fungus Ophiobolus disseminans, demonstrated considerable toxic effect with cell growth inhibition and subsequent cell death. Northern blot analysis showed a prominent 243-nt alpha-fragment of the 23S rRNA for the SL-2 toxin induction, thus confirming the efficient cleavage at the sarcin-ricin loop. Bioinformatic analysis of SL-2 led us to more sarcin-like candidates, which can be tested in the future. We then obtained a repertoire of four damaged RNA substrates: tRNAs cleaved at two positions, 30S ribosomal subunit damaged at the decoding center, and 70S ribosome damaged at SRL. We tested these four substrates with a candidate RtcB from each of the eight clusters to analyze repair activity in vitro. Preliminary results showed that candidates from clusters other than the ones mentioned above also demonstrate repair activity for different RNA substrates and may play a biological role in bacterial RNA repair.

Graduation Semester

2024-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2024 Adrika Raybarman

Owning Collections