Antibiotic inhibition of the movement of tRNA substrates through a peptidyl transferase cavity

1995 ◽  
Vol 73 (11-12) ◽  
pp. 877-885 ◽  
Author(s):  
Bo T. Porse ◽  
Cristina Rodriguez-Fonseca ◽  
Ilia Leviev ◽  
Roger A. Garrett
Keyword(s):  
Bond Formation ◽  
Ribosomal Subunit ◽  
Peptide Bond ◽  
Inhibitory Mechanism ◽  
Large Ribosomal Subunit ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Subunit Interactions ◽  
Antibiotic Inhibition ◽  
Peptidyl Transferase Centre

The present review attempts to deal with movement of tRNA substrates through the peptidyl transferase centre on the large ribosomal subunit and to explain how this movement is interrupted by antibiotics. It builds on the concept of hybrid tRNA states forming on ribosomes and on the observed movement of the 5′ end of P-site-bound tRNA relative to the ribosome that occurs on peptide bond formation. The 3′ ends of the tRNAs enter, and move through, a catalytic cavity where antibiotics are considered to act by at least three primary mechanisms: (i) they interfere with the entry of the aminoacyl moiety into the catalytic cavity before peptide bond formation; (ii) they inhibit movement of the nascent peptide along the peptide channel, a process that may generally involve destabilization of the peptidyl tRNA, and (iii) they prevent movement of the newly deacylated tRNA between the P/P and hybrid P/E sites on peptide bond formation.Key words: peptidyl transferase cavity, transient tRNA states, antibiotics, inhibitory mechanism, subunit–subunit interactions.

scholarly journals Ribosome crystallography: catalysis and evolution of peptide-bond formation, nascent chain elongation and its co-translational folding

2005 ◽  
Vol 33 (3) ◽  
pp. 488-492 ◽  
Author(s):  
A. Bashan ◽  
A. Yonath
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Trigger Factor ◽  
Chain Elongation ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Chemical Catalysis ◽  
Nascent Chain ◽  
Peptidyl Transferase Centre ◽  
Folding Space

A ribosome is a ribozyme polymerizing amino acids, exploiting positional- and substrate-mediated chemical catalysis. We showed that peptide-bond formation is facilitated by the ribosomal architectural frame, provided by a sizable symmetry-related region in and around the peptidyl transferase centre, suggesting that the ribosomal active site was evolved by gene fusion. Mobility in tunnel components is exploited for elongation arrest as well as for trafficking nascent proteins into the folding space bordered by the bacterial chaperone, namely the trigger factor.

A Possible Mechanism of Peptide Bond Formation on Ribosome without Mediation of Peptidyl Transferase

1999 ◽  
Vol 200 (2) ◽  
pp. 193-205 ◽  
Author(s):  
GOURAB KANTI DAS ◽  
DHANANJAY BHATTACHARYYA ◽  
DEBI PROSAD BURMA
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase

Ancient machinery embedded in the contemporary ribosome

2010 ◽  
Vol 38 (2) ◽  
pp. 422-427 ◽  
Author(s):  
Matthew J. Belousoff ◽  
Chen Davidovich ◽  
Ella Zimmerman ◽  
Yaron Caspi ◽  
Itai Wekselman ◽  
...  
Keyword(s):  
Gene Fusion ◽  
Bond Formation ◽  
Ribosomal Subunit ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Bacterial Organism ◽  
Or Gene ◽  
Definition Of ◽  
Folded Rna ◽  
Structural Definition

Structural analysis, supported by biochemical, mutagenesis and computational evidence, indicates that the peptidyltransferase centre of the contemporary ribosome is a universal symmetrical pocket composed solely of rRNA. This pocket seems to be a relic of the proto-ribosome, an ancient ribozyme, which was a dimeric RNA assembly formed from self-folded RNA chains of identical, similar or different sequences. This could have occurred spontaneously by gene duplication or gene fusion. This pocket-like entity was capable of autonomously catalysing various reactions, including peptide bond formation and non-coded or semi-coded amino acid polymerization. Efforts toward the structural definition of the early entity capable of genetic decoding involve the crystallization of the small ribosomal subunit of a bacterial organism harbouring a single functional rRNA operon.

On Ribosome Conservation and Evolution

2006 ◽  
Vol 52 (3-4) ◽  
pp. 359-374 ◽  
Author(s):  
Ilana Agmon ◽  
Anat Bashan ◽  
Ada Yonath
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Binding Modes ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Peptide Bonds ◽  
Peptidyl Transferase Center ◽  
Enzymatic Function ◽  
Rotatory Motion ◽  
Rna Fold

The ribosome is a ribozyme whose active site, the peptidyl transferase center (PTC), is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A-> P-site passage of the tRNA terminus in the peptidyl transferase center is performed by a rotatory motion, synchronized with the overall tRNA/mRNA sideways movement. Guided by the PTC, the rotatory motion leads to stereochemistry suitable for peptide bond formation, as well as for substrate-mediated catalysis, consistent with quantum mechanical calculations elucidating the transition state mechanism for peptide bond formation and indicating that the peptide bond is being formed during the rotatory motion. Analysis of substrate binding modes to inactive and active ribosomes illuminated the significant PTC mobility and supported the hypothesis that the ancient ribosome produced single peptide bonds and non-coded chains, utilizing free amino acids. Genetic control of the reaction evolved after poly-peptides capable of enzymatic function were created, and an ancient stable RNA fold was converted into tRNA molecules. As the symmetry relates only the backbone fold and nucleotide orientations, but not nucleotide sequence, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC has evolved by gene fusion, presumably by taking advantage of similar RNA fold structures.

A Competitive Model Reaction for the Ribosomal Peptide Bond Formation Catalyzed by Peptidyl Transferase

2014 ◽  
Vol 496-500 ◽  
pp. 17-20
Author(s):  
Lin Cheng ◽  
Nian Hong ◽  
Xiang Qun Xu ◽  
Jie Yang ◽  
You Quan Zhong
Keyword(s):  
Reaction Pathway ◽  
Bond Formation ◽  
Peptide Bond ◽  
Model Reaction ◽  
Reaction Pathways ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Reaction Field ◽  
Self Consistent ◽  
Competitive Model

In this work, a series of theoretical methods were employed to investigate the reaction mechanisms of ribosomal peptide bond formation catalyzed by peptidyl transferase. For the studies described in this paper, reaction pathways and free energy barriers for the model reaction of the peptide bond synthesis were studied by performing Ab initio calculation. Two self-consistent reaction field (SCRF) methods were used to calculate of the whole reaction pathway. These results show that the present theoretical reaction mechanism is a potential and competitive one for the reaction modeling of the ribosomal peptide synthesis.

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.

scholarly journals Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection

2020 ◽  
Vol 117 (7) ◽  
pp. 3610-3620 ◽  
Author(s):  
Justin C. Morse ◽  
Dylan Girodat ◽  
Benjamin J. Burnett ◽  
Mikael Holm ◽  
Roger B. Altman ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ternary Complex ◽  
Bond Formation ◽  
Critical Role ◽  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Elongation Factor Tu ◽  
Peptide Bond Formation ◽  
Gtp Hydrolysis

The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.

scholarly journals Madumycin II inhibits peptide bond formation by forcing the peptidyl transferase center into an inactive state

2017 ◽  
Vol 45 (12) ◽  
pp. 7507-7514 ◽  
Author(s):  
Ilya A. Osterman ◽  
Nelli F. Khabibullina ◽  
Ekaterina S. Komarova ◽  
Pavel Kasatsky ◽  
Victor G. Kartsev ◽  
...  
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Inactive State ◽  
Madumycin Ii
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