Investigating the Molecular Mechanism of Ribosome Recycling
| dc.contributor.advisor | Gagnon, Matthieu (magagnon@utmb.edu) | |
| dc.contributor.advisor | Yin, Yuhui (ywyin@utmb.edu) | |
| dc.contributor.committeeMember | Smith, Thomas (thosmith@utmb.edu) | |
| dc.contributor.committeeMember | Leiman, Petr (pgleiman@utmb.edu) | |
| dc.contributor.committeeMember | Chopra, Ashok (achopra@utmb.edu) | |
| dc.contributor.committeeMember | Tsai, Francis (ftsai@bcm.edu) | |
| dc.creator | Seely, Savannah Marie 1996- | |
| dc.creator.orcid | 0000-0001-7354-8913 | |
| dc.date.accessioned | 2024-04-08T12:07:17Z | |
| dc.date.available | 2024-04-08T12:07:17Z | |
| dc.date.created | 2024-05 | |
| dc.date.issued | May 2024 | |
| dc.date.submitted | May 2024 | |
| dc.date.updated | 2024-04-08T12:07:20Z | |
| dc.description.abstract | In all living cells, the ribosome translates the genetic information carried by messenger RNAs (mRNAs) into proteins. The process of ribosome recycling, a key step during protein synthesis that ensures ribosomal subunits remain available for new rounds of translation, has been largely overlooked. Despite being essential to the survival of the cell, several mechanistic aspects of ribosome recycling remain unclear. Aminoglycosides are a class of antibiotics that bind to ribosomal RNA and exert pleiotropic effects on ribosome function, including recycling inhibition. Amikacin, the semisynthetic derivative of kanamycin, is commonly used for treating severe infections with multidrug-resistant, aerobic Gram-negative bacteria. Amikacin carries the 4-amino-2-hydroxy butyrate (AHB) moiety at the N1 amino group of the central 2-deoxystreptamine (2-DOS) ring, which may confer amikacin a unique ribosome inhibition profile. During stress conditions such as antibiotic exposure, ribosomes stall on messenger RNAs, leading to inhibition of protein synthesis. To remobilize ribosomes, bacteria use rescue factors such as HflXr, that catalyzes the dissociation of translationally inactive ribosomes into individual subunits. Here we use in vitro fast kinetics combined with X-ray crystallography and cryo-EM to dissect the mechanisms of ribosome inhibition by amikacin and the rescue of stalled ribosome through HflXr-mediated recycling. Amikacin interferes with tRNA translocation, release factor-mediated peptidyl-tRNA hydrolysis, and ribosome recycling, traits attributed to the additional interactions amikacin makes with the decoding center. The binding site in the large ribosomal subunit proximal to the 3’-end of tRNA in the peptidyl (P) site lays the groundwork for rational design of amikacin derivatives with improved antibacterial properties. Using time-resolved cryo-EM, we show that within the 70S ribosome, HflXr displaces helix H69 of the 50S subunit and induces long-range movements of the platform domain of the 30S subunit, disrupting inter-subunit bridges B2b, B2c, B4, B7a, and B7b. Our findings unveil a unique ribosome recycling strategy by HflXr which is distinct from that mediated by RRF and EF-G. The resemblance between HflXr and housekeeping HflX suggests that the alternative ribosome recycling mechanism reported is universal in the prokaryotic kingdom. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | ||
| dc.identifier.uri | https://hdl.handle.net/2152.3/12384 | |
| dc.language.iso | English | |
| dc.subject.other | Structural Biology, Cryo-EM, Ribosome, Protein Synthesis, Antibiotics | |
| dc.title | Investigating the Molecular Mechanism of Ribosome Recycling | |
| dc.type | Thesis | |
| dc.type.material | text | |
| thesis.degree.college | UTMB Graduate School of Biomedical Sciences | |
| thesis.degree.department | Biochemistry, Cell, and Molecular Biology | |
| thesis.degree.discipline | Structural Biology and Biophysics | |
| thesis.degree.grantor | The University of Texas Medical Branch at Galveston | |
| thesis.degree.name | Biochemistry and Molecular Biology (Doctoral) | |
| thesis.degree.school | University of Texas Medical Branch |