scholarly journals Proline codon pair selection determines ribosome pausing strength and translation efficiency in bacteria

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Ralph Krafczyk ◽  
Fei Qi ◽  
Alina Sieber ◽  
Judith Mehler ◽  
Kirsten Jung ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mrna Level ◽  
Mrna Translation ◽  
Peptide Bond ◽  
Translation Efficiency ◽  
Peptide Bond Formation ◽  
Copy Numbers ◽  
Nascent Chain ◽  
Ribosome Pausing ◽  
Codon Pairs

AbstractThe speed of mRNA translation depends in part on the amino acid to be incorporated into the nascent chain. Peptide bond formation is especially slow with proline and two adjacent prolines can even cause ribosome stalling. While previous studies focused on how the amino acid context of a Pro-Pro motif determines the stalling strength, we extend this question to the mRNA level. Bioinformatics analysis of the Escherichia coli genome revealed significantly differing codon usage between single and consecutive prolines. We therefore developed a luminescence reporter to detect ribosome pausing in living cells, enabling us to dissect the roles of codon choice and tRNA selection as well as to explain the genome scale observations. Specifically, we found a strong selective pressure against CCC/U-C, a sequon causing ribosomal frameshifting even under wild-type conditions. On the other hand, translation efficiency as positive evolutionary driving force led to an overrepresentation of CCG. This codon is not only translated the fastest, but the corresponding prolyl-tRNA reaches almost saturating levels. By contrast, CCA, for which the cognate prolyl-tRNA amounts are limiting, is used to regulate pausing strength. Thus, codon selection both in discrete positions but especially in proline codon pairs can tune protein copy numbers.

Eco-Friendly Synthesis of Peptides Using Fmoc-Amino Acid Chlorides as Coupling Agent Under Biphasic Condition

2020 ◽  
Vol 27 ◽  
Author(s):  
Santosh Y. Khatavi ◽  
K. Kantharaju
Keyword(s):  
Amino Acid ◽  
Acid Chloride ◽  
Acid Ester ◽  
Analytical Techniques ◽  
Peptide Bond ◽  
Amino Acid Ester ◽  
Acid Chlorides ◽  
Peptide Bond Formation ◽  
Green Protocol ◽  
Additional Base

Background: Agro-waste derived solvent media act as a greener process for the peptide bond formation using Nα - Fmoc-amino acid chloride and amino acid ester salt with in situ neutralization and coupling under biphasic condition. The Fmoc-amino acid chlorides are prepared by the reported procedure of freshly distilled SOCl2 with dry CH2Cl2. The protocol found many added advantages such as neutralization of amino acid ester salt and not required additional base for the neutralization, and directly coupling take place with Fmoc-amino acid chloride gave final product dipeptide ester in good to excellent yields. The protocol occurs with complete stereo chemical integrity of the configuration of substrates. Here, we revisited Schotten-Baumann condition, instead of using inorganic base. Objective: To develop green protocol for the synthesis of peptide bond using Fmoc-amino acid chloride with amino acid esters salt. Methods: The final product isolated is analyzed in several spectroscopic and analytical techniques such as FT-IR, 1H-, 13CNMR, Mass spectrometry and RP-HPLC to check stereo integrity and purity of the product. Conclusion: The present method developed greener using natural agro-waste (lemon fruit shell ash) derived solvent medium for the reaction and not required chemical entity.

Oxidative Peptide Bond Formation of Glycine-Amino Acid using 2-(Aminomethyl)malononitrile as a Glycine Unit

2021 ◽  
Author(s):  
Xiaoling Wang ◽  
Jing Li ◽  
Yujiro Hayashi
Keyword(s):  
Aqueous Solution ◽  
Amino Acid ◽  
Bond Formation ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Amide Linkage

Amide linkage of glycine-amino acid was synthesized by coupling of substituted 2-(aminomethyl)malononitrile as a C-terminal glycine unit and N-terminal amine using CsOAc and O2 in aqueous solution. This is a...

scholarly journals Ribosome elongation kinetics of consecutively charged residues are coupled to electrostatic force

2021 ◽  
Author(s):  
Sarah E Leininger ◽  
Judith Rodriguez ◽  
Quyen V Vu ◽  
Yang Jiang ◽  
Ma Suan Li ◽  
...  
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Electrostatic Force ◽  
Ribosome Profiling ◽  
Peptide Bond Formation ◽  
Translation Speed ◽  
Charged Residues ◽  
Nascent Chain ◽  
Chain Conformations

The speed of protein synthesis can dramatically change when consecutively charged residues are incorporated into an elongating nascent protein by the ribosome. The molecular origins of this class of allosteric coupling remain unknown. We demonstrate, using multi-scale simulations, that positively charged residues generate large forces that pull the P-site amino acid away from the A-site amino acid. Negatively charged residues generate forces of similar magnitude but opposite direction. And that these conformational changes, respectively, raise and lower the transition state barrier height to peptide bond formation, explaining how charged residues mechanochemically alter translation speed. This mechanochemical mechanism is consistent with in vivo ribosome profiling data exhibiting a proportionality between translation speed and the number of charged residues, experimental data characterizing nascent chain conformations, and a previously published cryo-EM structure of a ribosome-nascent chain complex containing consecutive lysines. These results expand the role of mechanochemistry in translation, and provide a framework for interpreting experimental results on translation speed.

scholarly journals Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics

2021 ◽  
Author(s):  
Kaitlyn Tsai ◽  
Vanja Stojković ◽  
D. John Lee ◽  
Iris D. Young ◽  
Teresa Szal ◽  
...  
Keyword(s):  
Peptide Bond ◽  
Structural Basis ◽  
Side Chain ◽  
Specific Inhibition ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Nascent Chain ◽  
Context Specific ◽  
Small Hydrophobic ◽  
Penultimate Position

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. In this study, we determined that the second-generation oxazolidinone radezolid also induces stalling with alanine at the penultimate position. However, the molecular basis for context-specificity of these inhibitors has not been elucidated. In this study, we determined high-resolution cryo-EM structures of both linezolid and radezolid-stalled ribosome complexes. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling by forcing the antibiotic to adopt a conformation that narrows the hydrophobic alanine pocket. Together, the structural and biochemical findings presented in this work provide molecular understanding of context-specific inhibition of translation by clinically important oxazolidinone antibiotics.

scholarly journals Short translational ramp determines efficiency of protein synthesis

2019 ◽  
Author(s):  
Manasvi Verma ◽  
Junhong Choi ◽  
Kyle A. Cottrell ◽  
Zeno Lavagnino ◽  
Erica N. Thomas ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Single Molecule ◽  
Translation Initiation ◽  
Protein Translation ◽  
Translational Efficiency ◽  
Translation Efficiency ◽  
Sequence Motifs ◽  
Peptide Bond Formation

AbstractIt is generally assumed that translation efficiency is governed by translation initiation. However, the efficiency of protein synthesis is regulated by multiple factors including tRNA abundance, codon composition, mRNA motifs and amino-acid sequence1–4. These factors influence the rate of protein synthesis beyond the initiation phase of translation, typically by modulating the rate of peptide-bond formation and to a lesser extent that of translocation. The slowdown in translation during the early elongation phase, known as the 5’ translational ramp, likely contributes to the efficiency of protein synthesis 5–9. Multiple mechanisms, which could explain the molecular basis for this translational ramp, have been proposed that include tRNA abundance bias6,9, the rate of translation initiation10–15, mRNA and ribosome structure 11,12,14,16–18, or retention of initiation factors during early elongation events 19. Here, we show that the amount of synthesized protein (translation efficiency) depends on a short translational ramp that comprises the first 5 codons in mRNA. Using a library of more than 250,000 reporter sequences combined with in vitro and in vivo protein expression assays, we show that differences in the short ramp can lead to 3 to 4 orders of magnitude changes in protein abundance. The observed difference is not dependent on tRNA abundance, efficiency of translation initiation, or overall mRNA structure. Instead, we show that translation is regulated by amino-acid-sequence composition and local mRNA sequence. Single-molecule measurements of translation kinetics indicate substantial pausing of ribosome and abortion of protein synthesis on the 4th or 5th codon for distinct amino acid or nucleotide compositions. Introduction of preferred sequence motifs, only at the exact positions within the mRNA, improves protein synthesis for recombinant proteins, indicating an evolutionarily conserved mechanism for controlling translational efficiency.

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.

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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