scholarly journals Mechanism of ribosome rescue by ArfA and RF2

2016 ◽  
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

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.

scholarly journals Mechanism of ribosome rescue by ArfA and RF2

eLife ◽  
2017 ◽  
Vol 6 ◽  
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.

scholarly journals Conformational control of translation termination on the 70S ribosome

2017 ◽  
Author(s):  
Egor Svidritskiy ◽  
Andrei A. Korostelev
Keyword(s):  
Conformational Changes ◽  
Catalytic Domain ◽  
Stop Codon ◽  
Translation Termination ◽  
Release Factor ◽  
Codon Recognition ◽  
Peptide Release ◽  
Stop Codon Recognition ◽  
70S Ribosome ◽  
A Site

AbstractTranslation termination ensures proper lengths of cellular proteins. During termination, release factor (RF) recognizes a stop codon and catalyzes peptide release. Conformational changes in RF are thought to underlie accurate translation termination. If true, the release factor should bind the A-site codon in inactive (compact) conformation(s), but structural studies of ribosome termination complexes have only captured RFs in an extended, active conformation. Here, we identify a hyper-accurate RF1 variant, and present crystal structures of 70S termination complexes that suggest a structural pathway for RF1 activation. In the presence of blasticidin S, the catalytic domain of RF1 is removed from the peptidyl-transferase center, whereas the codon-recognition domain is fully engaged in stop-codon recognition in the decoding center. RF1 codon recognition induces decoding-center rearrangements that precede accommodation of the catalytic domain. Our findings suggest how structural dynamics of RF1 and the ribosome coordinate stop-codon recognition with peptide release, ensuring accurate translation termination.

scholarly journals Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release

2018 ◽  
Vol 115 (3) ◽  
pp. E382-E389 ◽  
Author(s):  
Thomas Philipp Hoernes ◽  
Nina Clementi ◽  
Michael Andreas Juen ◽  
Xinying Shi ◽  
Klaus Faserl ◽  
...  
Keyword(s):  
Stop Codon ◽  
Release Factor ◽  
Stop Codons ◽  
Codon Recognition ◽  
Chemically Modified ◽  
Peptide Release ◽  
Stop Codon Recognition ◽  
A Site ◽  
Chemical Groups ◽  
Insight Into

Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons.

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

2016 ◽  
Vol 113 (18) ◽  
pp. 4994-4999 ◽  
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.

scholarly journals Histidine 197 in Release Factor 1 Is Essential for A Site Binding and Peptide Release

Biochemistry ◽  
2010 ◽  
Vol 49 (43) ◽  
pp. 9385-9390 ◽  
Author(s):  
Andrew Field ◽  
Byron Hetrick ◽  
Merrill Mathew ◽  
Simpson Joseph
Keyword(s):  
Release Factor ◽  
Peptide Release ◽  
A Site ◽  
Site Binding

Translational termination efficiency in both bacteria and mammals is regulated by the base following the stop codon

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1095-1103 ◽  
Author(s):  
Warren P. Tate ◽  
Elizabeth S. Poole ◽  
Julie A. Horsfield ◽  
Sally A. Mannering ◽  
Chris M. Brown ◽  
...  
Keyword(s):  
Stop Codon ◽  
Stop Signal ◽  
Physical Contact ◽  
Release Factor ◽  
Wide Range ◽  
Highly Expressed Genes ◽  
Peptidyltransferase Center ◽  
A Site ◽  
Translational Termination

The translational stop signal and polypeptide release factor (RF) complexed with Escherichia coli ribosomes have been shown to be in close physical contact by site-directed photochemical cross-linking experiments. The RF has a protease-sensitive site in a highly conserved exposed loop that is proposed to interact with the peptidyltransferase center of the ribosome. Loss of peptidyl–tRNA hydrolysis activity and enhanced codon–ribosome binding by the cleaved RF is consistent with a model whereby the RF spans the decoding and peptidyltransferase centers of the ribosome with domains of the RF linked by conformational coupling. The cross-link between the stop signal and RF at the ribosomal decoding site is influenced by the base following the termination codon. This base determines the efficiency with which the stop signal is decoded by the RF in both mammalian and bacterial systems in vivo. The wide range of efficiencies correlates with the frequency with which the signals occur at natural termination sites, with rarely used weak signals often found at recoding sites and strong signals found in highly expressed genes. Stop signals are found at some recoding sites in viruses where −1 frame-shifting occurs, but the generally accepted mechanism of simultaneous slippage from the A and P sites does not explain their presence here. The HIV-1 gag-pol −1 frame shifting site has been used to show that stop signals significantly influence frame-shifting efficiency on prokaryotic ribosomes by a RF-mediated mechanism. These data can be explained by an E/P site simultaneous slippage mechanism whereby the stop codon actually enters the ribosomal A site and can influence the event.Key words: translational stop signal, decoding, release factor, frame-shifting.

scholarly journals The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center

2015 ◽  
Vol 112 (19) ◽  
pp. 6038-6043 ◽  
Author(s):  
Michael T. Englander ◽  
Joshua L. Avins ◽  
Rachel C. Fleisher ◽  
Bo Liu ◽  
Philip R. Effraim ◽  
...  
Keyword(s):  
Amino Acids ◽  
Physiological Role ◽  
Peptidyl Transferase ◽  
Translational Machinery ◽  
Peptidyl Transferase Center ◽  
Peptidyl Transfer ◽  
Site Use ◽  
Dynamics Simulations ◽  
A Site ◽  
Trna Binding

The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells.

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

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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