Elucidating the molecular mechanisms of ribosome rescue and repair by bacterial PrfH and RtcB2

Tian, Yannan

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https://hdl.handle.net/2142/125779 Copy

Description

Title

Elucidating the molecular mechanisms of ribosome rescue and repair by bacterial PrfH and RtcB2

Author(s)

Tian, Yannan

Issue Date

2024-07-10

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

Huang, Raven

Doctoral Committee Chair(s)

Huang, Raven

Committee Member(s)

• van der Donk , Wilfred A.
• Jin, Hong
• Stadtmueller , Beth

Department of Study

Biochemistry

Discipline

Biochemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

RtcB2 PrfH

Abstract

Conflicts between organisms are fundamental biological phenomena. In an environment of limited nutrition, some bacteria produce toxins to kill their neighbors to eliminate competition. Since the protein translation machinery is essential and universally conserved in all organisms, it is the main target of both small-molecule and protein toxins. Most protein toxins targeting protein translation are ribotoxins, inflicting damage on essential RNAs required for protein translation. In order to survive, many organisms have developed RNA repair systems, which can heal and seal the cleaved RNA molecules to restore their functions. In E. coli, RtcB, the founding member of RtcB family, is an RNA ligase. We found that most E. coli strains have a second RtcB (named RtcB2) encoded in a rtcB-prfH operon. Because PrfH (protein release factor homology) is likely to interact with the ribosome, we hypothesized that RtcB2-PrfH pair are likely to be engaged in ribosome rescue and repair. Therefore, we carried out a series biochemical studies of RtcB2-PrfH from E. coli together with the ribosome damaged in the decoding center. Our peptide release assays revealed that PrfH releases the peptide attached to P-site tRNA only from the damaged 70S ribosome, not the intact ribosome. The cryo-EM structure of EcPrfH in complex with the damaged 70S ribosome provides molecular insight into EcPrfH recognizing the ribosomal damage site. In vitro RNA repair assays showed that EcRtcB2 specifically repairs the damaged 30S ribosome subunit, not the damaged tRNAs. In addition to EcPrfH, we also studied PrfH from Capnocytophaga gingivalis (CgPrfH). We found that, compared to EcPrfH, CgPrfH has an additional C-terminal tail. This led us to perform a systematic bioinformatic analysis of PrfH protein family, revealing that there are two subfamilies of PrfH exemplified by EcPrfH and CgPrfH. In vitro peptide release assays using recombinant CgPrfH revealed that, unlike EcPrfH, CgPrfH can release the P-site peptide in both damaged and intact ribosome. Cryo-EM structures of CgPrfH in complex with the damaged and intact ribosome provide molecular insight into ribosome recognition. The structure of CgPrfH in complex with the damaged ribosome revealed that the C-terminal tail is unstructured, and the damaged ribosome is recognized by the ribosome recognition domain (RRD) similar as the one found in EcPrfH. The structure of CgPrfH in complex with the intact ribosome, however, showed that RRD has limited contact with the ribosome. Instead, the C-terminal tail, occupying the empty mRNA channel, is mainly responsible for the recognition of the intact ribosome. Thus, CgPrfH plays dual biological functions, employing different regions to recognize two different stalled ribosomes for rescue. Although we have shown that RtcB2 is able to repair the damaged 30S ribosome via in vitro biochemical assays, how the damaged 30S ribosome subunit is recognized by RtcB2, as well as the repair mechanism, remains unknown. Therefore, we carried out cryo-EM studies of EcRtcB2 in complex with the damaged 30S ribosome subunit. Two high-resolution cryo-EM maps were obtained, corresponding to EcRtcB2 in complex with the repaired 30S subunit and the damaged 30S subunit alone, respectively. Structural analysis provides molecular insight into how EcRtcB2, but not EcRtcB1, recognizing the 30S ribosome subunit. The results we have clearly demonstrate that the RtcB2-PrfH pair is the first ribosomal RNA repair system that specifically rescues and repairs stalled ribosomes cleaved between A1493 and G1494 within the decoding center.

Graduation Semester

2024-08

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/125779

Copyright and License Information

closed access: a lot of work described in my dissertation has not been published yet, and we are still working on them, so I want to keep closed access for my dissertation

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