scholarly journals Visualization of Trna Movements on the Escherichia coli 70s Ribosome during the Elongation Cycle

2000 ◽  
Vol 150 (3) ◽  
pp. 447-460 ◽  
Author(s):  
Rajendra K. Agrawal ◽  
Christian M.T. Spahn ◽  
Pawel Penczek ◽  
Robert A. Grassucci ◽  
Knud H. Nierhaus ◽  
...  
Keyword(s):  
Bond Formation ◽  
Three Dimensional ◽  
Peptide Bond ◽  
High Accuracy ◽  
Peptide Bond Formation ◽  
Elongation Cycle ◽  
Before And After ◽  
70S Ribosome ◽  
Similar Accuracy ◽  
A Site

Three-dimensional cryomaps have been reconstructed for tRNA–ribosome complexes in pre- and posttranslocational states at 17-Å resolution. The positions of tRNAs in the A and P sites in the pretranslocational complexes and in the P and E sites in the posttranslocational complexes have been determined. Of these, the P-site tRNA position is the same as seen earlier in the initiation-like fMet-tRNAfMet-ribosome complex, where it was visualized with high accuracy. Now, the positions of the A- and E-site tRNAs are determined with similar accuracy. The positions of the CCA end of the tRNAs at the A site are different before and after peptide bond formation. The relative positions of anticodons of P- and E-site tRNAs in the posttranslocational state are such that a codon–anticodon interaction at the E site appears feasible.

ONIOM and ab-initio calculations on the mechanism of uncatalyzed peptide bond formation

2012 ◽  
Vol 90 (6) ◽  
pp. 691-700 ◽  
Author(s):  
Hadieh Monajemi ◽  
Mohammad Noh Daud ◽  
Sharifuddin Mohd. Zain ◽  
Wan Ahmad Tajuddin Wan Abdullah
Keyword(s):  
Amino Acids ◽  
Bond Formation ◽  
Computational Cost ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Initiator Trna ◽  
Reduced Rate ◽  
Surface Scan ◽  
A Site

Finding a proper transition structure for the peptide bond formation process can lead one to a better understanding of the role of ribosome in catalyzing this reaction. Using computer simulations, we performed the potential energy surface scan on the ester bond dissociation of P-site aminoacyl-tRNA and the peptide bond formation of P-site and A-site amino acids. The full fragments of initiator tRNAimet and elongator tRNAphe are attached to both cognate and non-cognate amino acids as the P-site substrate. The A-site amino acid for all four calculations is methionine. We used ONIOM calculations to reduce the computational cost. Our study illustrates the reduced rate of peptide bond formation for misacylated tRNAimet in the absence of ribosomal bases. The misacylated elongator tRNAphe, however, did not show any difference in its PES compared with that for the phe-tRNAphe. This demonstrates the structural specification of initiator tRNAimet for the amino acids side chain.

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 Disome-seq reveals widespread ribosome collisions that recruit co-translational chaperones

2019 ◽  
Author(s):  
Taolan Zhao ◽  
Yan-Ming Chen ◽  
Yu Li ◽  
Jia Wang ◽  
Siyu Chen ◽  
...  
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Protein Homeostasis ◽  
Peptide Bond Formation ◽  
Translation Elongation ◽  
Protein Levels ◽  
Cryo Electron Microscopy ◽  
Exit Tunnel ◽  
Hidden Layer ◽  
A Site

ABSTRACTRegulation of translation elongation plays a crucial role in determining absolute protein levels and ensuring the correct localization and folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes (ribo-seq). However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation. In this study, we performed disome-seq to sequence mRNA fragments protected by two stacked ribosomes — a product of translational pauses during which the 5′-ribosome collides with the 3′-paused one. We detected widespread ribosome collisions that are missed in traditional ribo-seq. These collisions are due to 1) slow ribosome release when stop codons are at the A-site, 2) slow peptide bond formation from proline, glycine, asparagine, and cysteine when they are at the P-site, and 3) slow leaving of polylysine from the exit tunnel of ribosomes. The paused ribosomes can continue translating after collisions, as suggested by the structure of disomes obtained by cryo-electron microscopy (cryo-EM). Collided ribosomes recruit chaperones, which can aid in the co-translational folding of the nascent peptides. Therefore, cells use regulated ribosome collisions to ensure protein homeostasis.

scholarly journals Insights into the base-pairing preferences of 8-oxoguanosine on the ribosome

2019 ◽  
Vol 47 (18) ◽  
pp. 9857-9870 ◽  
Author(s):  
Erica N Thomas ◽  
Carrie L Simms ◽  
Hannah E Keedy ◽  
Hani S Zaher
Keyword(s):  
Base Pair ◽  
Structural Changes ◽  
Bond Formation ◽  
Peptide Bond ◽  
Underlying Mechanism ◽  
Peptide Bond Formation ◽  
Base Pairing ◽  
Base Pairs ◽  
Free Dna ◽  
A Site

Abstract Of the four bases, guanine is the most susceptible to oxidation, which results in the formation of 8-oxoguanine (8-oxoG). In protein-free DNA, 8-oxodG adopts the syn conformation more frequently than the anti one. In the syn conformation, 8-oxodG base pairs with dA. The equilibrium between the anti and syn conformations of the adduct are known to be altered by the enzyme recognizing 8-oxodG. We previously showed that 8-oxoG in mRNA severely disrupts tRNA selection, but the underlying mechanism for these effects was not addressed. Here, we use miscoding antibiotics and ribosome mutants to probe how 8-oxoG interacts with the tRNA anticodon in the decoding center. Addition of antibiotics and introduction of error-inducing mutations partially suppressed the effects of 8-oxoG. Under these conditions, rates and/or endpoints of peptide-bond formation for the cognate (8-oxoG•C) and near-cognate (8-oxoG•A) aminoacyl-tRNAs increased. In contrast, the antibiotics had little effect on other mismatches, suggesting that the lesion restricts the nucleotide from forming other interactions. Our findings suggest that 8-oxoG predominantly adopts the syn conformation in the A site. However, its ability to base pair with adenosine in this conformation is not sufficient to promote the necessary structural changes for tRNA selection to proceed.

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