Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/65648
Title: Structural study of ribosome : 1) structural basis of EF-G in translocation. 2) molecular mechanism of ribosome-dependent toxin YoeB in mRNA cleavage
Authors: Chen, Yun
Keywords: DRNTU::Science::Biological sciences::Biophysics
Issue Date: 2015
Source: Chen, Y. (2015). Structural study of ribosome : 1) structural basis of EF-G in translocation. 2) molecular mechanism of ribosome-dependent toxin YoeB in mRNA cleavage. Doctoral thesis, Nanyang Technological University, Singapore.
Abstract: During bacterial protein synthesis, elongation factor G (EF-G) facilitates the forward movement of the tRNA–mRNA by one codon, which is coupled to the ratchet-like movement of the ribosome and triggered by EF-G–mediated GTP hydrolysis. This process, known as translocation, is the most conserved part between prokaryotic and eukaryotic translation. Despite its significance and decades of studies, the detailed mechanism of translocation still largely remains elusive. In current thesis, we present the structure of the Thermus thermophilus ribosome in complex with EF-G trapped by a GTP analogue. Binding with the ribosome and the GTP analogue facilitate the stabilization of EF-G switch I region, resulting in an ordered model of the active site. The positioning of the catalytic His87 into the active site is coupled to the hydrophobic-gate opening and involves the ribosomal 23S rRNA sarcin-ricin loop as well as domain III of EF-G. These findings provide a structural basis for the GTPase activation of EF-G, based on which, a substrate-promoted mechanism is proposed. The structure also reveals that the entire L1 stalk adopts a completely closed conformation by shifting toward the 50S body, thereby forming extensive contacts with the hybrid P/E tRNA. Together with the newly established interactions of the P/E tRNA with ribosomal proteins S13 and S19, these findings shed light on how the formation and stabilization of the hybrid tRNA is coupled to the head swiveling and body rotation of the 30S subunit, as well as to the closure of the L1 stalk of the 50S subunit. Toxin-antitoxin (TA) modules, composed of a stable toxin and its cognate liable antitoxin, are common in prokaryotes. YoeB, a typical endoribonuclease, belongs to the YoeB-YefM TA module and mediates cellular adaptation in diverse bacteria by degrading mRNAs upon its activation. While the catalytic core of YoeB is thought to be identical to other well-studied nucleases, this enzyme specifically targets mRNA substrates that are associated with ribosomes in vivo. However, the molecular mechanism of mRNA recognition and cleavage by YoeB, and the requirement of ribosome for its optimal activity, largely remain elusive. Here, we present the structure of YoeB bound to the 70S ribosome in a pre-cleavage state, revealing that both 30S and 50S subunits participate in YoeB binding. The mRNA substrate is recognized by the catalytic core of YoeB. The general base/acid (Glu46/His83) are within hydrogen-bonding distance to their reaction atoms, demonstrating an active conformation of YoeB on ribosome. Moreover, the orientation of mRNA involves the universally conserved A1493 and G530 of 16S rRNA. In addition, mass spectrometry data indicated that YoeB cleaves mRNA following the second position at the A-site codon, resulting in a final product with a 3′–phosphate at the newly formed 3′ end. Our results demonstrate a classical acid-base catalysis for YoeB–mediated RNA hydrolysis and provide an insight into how the ribosome is essential for its specific activity.
URI: https://hdl.handle.net/10356/65648
DOI: 10.32657/10356/65648
Fulltext Permission: open
Fulltext Availability: With Fulltext
Appears in Collections:SBS Theses

Files in This Item:
File Description SizeFormat 
Thesis_1st_amendment_ChenYun_GYG.pdf22.68 MBAdobe PDFThumbnail
View/Open

Page view(s)

249
Updated on May 9, 2021

Download(s) 50

78
Updated on May 9, 2021

Google ScholarTM

Check

Altmetric


Plumx

Items in DR-NTU are protected by copyright, with all rights reserved, unless otherwise indicated.