Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

AbstractArfA rescues ribosomes stalled on truncated mRNAs by recruiting the release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2-Å resolution cryo-EM structures – determined from a single sample – of the 70S ribosome with ArfA∙RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation, hitherto unobserved in 70S termination complexes. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA “senses” the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

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Abstract P-29: Cryoem Study of the Inhibition of Bacterial Ribosomes by Madumycin II

International Journal of Biomedicine ◽

10.21103/ijbm.11.suppl_1.p29 ◽

2021 ◽

Vol 11 (Suppl_1) ◽

pp. S24-S25

Author(s):

Alena Yakusheva ◽

Olga Shulenina ◽

Evgeny Pichkur ◽

Alena Paleskava ◽

Alexander Myasnikov ◽

...

Keyword(s):

Amino Acid ◽

High Resolution ◽

Bond Formation ◽

Protein Translation ◽

Peptide Bond ◽

Peptide Bond Formation ◽

Peptidyl Transferase ◽

Peptidyl Transferase Center ◽

70S Ribosome ◽

Madumycin Ii

Background: The efficiency of widely used antibiotics is limited by continuous improvement of resistance mechanisms. Thus, the research of poorly studied drugs that have not received practical use until now becomes relevant again. Protein translation is one of the major targets for antibiotics. Madumycin II (MADU) is an antibiotic of the streptogramin A class that binds to the peptidyl transferase center of the initiated bacterial 70S ribosome inhibiting the ﬁrst cycle of peptide bond formation (I.A. Osterman et al. Nucleic Acids Res., 2017). The ability of MADU to interfere with translating ribosome is an open question that we address by investigation of high-resolution cryo-EM structures of MADU bound 70S ribosome complexes from Escherichia coli. Methods: Purified initiated and translating ribosome complexes preincubated with MADU were applied onto freshly glow discharged carbon-coated grids (Quantifoil R 1.2/1.3) and flash-frozen in the liquid ethane pre-cooled by liquid nitrogen in the Vitrobot Mark IV. Frozen grids were transferred into an in-house Titan Krios microscope. Data were collected using EPU software. Movie stacks were preprocessed in Warp software. For image processing, we have used several software packages: Relion 3.1, CryoSPARC, and CisTEM. The model was built in Coot. Results: We have obtained high-resolution cryo-EM structures of two ribosomal complexes with MADU before and after the ﬁrst cycle of peptide bond formation with an average resolution of 2.3 Å. Preliminary analysis of the structures shows no major differences in the MADU binding mode to the ribosomal complexes under study suggesting that the quantity of amino acid residues attached to the P-site tRNA does not impact MADU bonding. Moreover, in both cases, we observed similar destabilization of the CCA-ends of A- and P-site tRNAs underlining the comparable influence of MADU on the ribosomal complexes. Conclusion: Our results suggest that although MADU binding site is located in the peptidyl transferase center, the presence of the second amino acid residue on the P-site tRNA does not preclude antibiotic binding. We assume that further elongation of the polypeptide chain would not have any impact either. High conformational lability of the CCA-ends of tRNA at the A and P sites upon binding of MADU obviously plays an important role in the inhibition mechanism of the bacterial ribosome. The further structural and biochemical analysis will be necessary to shed more light on the detailed mechanism of MADU action.

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Elongation factor 4 remodels the A-site tRNA on the ribosome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1522932113 ◽

2016 ◽

Vol 113 (18) ◽

pp. 4994-4999 ◽

Cited By ~ 13

Author(s):

Matthieu G. Gagnon ◽

Jinzhong Lin ◽

Thomas A. Steitz

Keyword(s):

Crystal Structure ◽

Conformational Changes ◽

Thermus Thermophilus ◽

Elongation Factor ◽

Peptidyl Transferase ◽

Peptidyl Transferase Center ◽

70S Ribosome ◽

Regulate Protein ◽

A Site ◽

Hydrolysis Of

During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5′-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant.

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Cross-crystal averaging reveals that the structure of the peptidyl-transferase center is the same in the 70S ribosome and the 50S subunit

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0711076105 ◽

2008 ◽

Vol 105 (2) ◽

pp. 500-505 ◽

Cited By ~ 16

Author(s):

M. Simonovic ◽

T. A. Steitz

Keyword(s):

Peptidyl Transferase ◽

Peptidyl Transferase Center ◽

70S Ribosome

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Mechanism of ribosome rescue by ArfA and RF2

eLife ◽

10.7554/elife.23687 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 27

Author(s):

Gabriel Demo ◽

Egor Svidritskiy ◽

Rohini Madireddy ◽

Ruben Diaz-Avalos ◽

Timothy Grant ◽

...

Keyword(s):

Structural Dynamics ◽

Stop Codon ◽

Release Factor ◽

Peptidyl Transferase ◽

Peptidyl Transferase Center ◽

C Terminus ◽

Peptide Release ◽

N Terminus ◽

70S Ribosome ◽

A Site

ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2 Å resolution cryo-EM structures – determined from a single sample – of the 70S ribosome with ArfA•RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA ‘senses’ the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

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