scholarly journals Rqc1 and other yeast proteins containing highly positively charged sequences are not targets of the RQC complex

2019 ◽  
Author(s):  
Géssica C. Barros ◽  
Rodrigo D. Requião ◽  
Rodolfo L. Carneiro ◽  
Claudio A. Masuda ◽  
Mariana H. Moreira ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Translation Termination ◽  
Ribosome Profiling ◽  
Translation Efficiency ◽  
Ribosome Stalling ◽  
Reporter Systems ◽  
Exit Tunnel ◽  
Endogenous Proteins ◽  
Ribosome Exit Tunnel ◽  
Positively Charged

ABSTRACTHighly positively charged protein segments are known to result in poor translation efficiency. This effect is explained by ribosome stalling caused by electrostatic interactions between the nascent peptide and the negatively charged ribosome exit tunnel, leading to translation termination followed by protein degradation mediated by the RQC complex. These polybasic sequences are mainly studied in the context of artificial reporter systems. Examples of endogenous yeast proteins targeted by the RQC complex are Rqc1, a protein essential for RQC function, and Sdd1. Both contain polybasic sequences that are thought to activate the RQC, leading to protein down-regulation. Here, we investigated whether the RQC complex regulates other endogenous proteins with polybasic sequences. We show by bioinformatics, ribosome profiling data analysis, and western blot that endogenous proteins containing polybasic sequences similar to, or even more positively charged than those of Rqc1 and Sdd1, are not targeted by the RQC complex suggesting that endogenous polybasic sequences are not sufficient to induce this type of regulation. Finally, our results also suggest that Rqc1 is regulated post-translationally by the E3 component of the RQC complex Ltn1, in a manner independent of the RQC complex.

Influence of nascent polypeptide positive charges on translation dynamics

2020 ◽  
Vol 477 (15) ◽  
pp. 2921-2934
Author(s):  
Rodrigo D. Requião ◽  
Géssica C. Barros ◽  
Tatiana Domitrovic ◽  
Fernando L. Palhano
Keyword(s):  
Mrna Decay ◽  
Translation Termination ◽  
Published Data ◽  
Translation Efficiency ◽  
Amino Acid Residues ◽  
High Concentration ◽  
Strong Argument ◽  
Translation Arrest ◽  
Exit Tunnel ◽  
Positively Charged

Protein segments with a high concentration of positively charged amino acid residues are often used in reporter constructs designed to activate ribosomal mRNA/protein decay pathways, such as those involving nonstop mRNA decay (NSD), no-go mRNA decay (NGD) and the ribosome quality control (RQC) complex. It has been proposed that the electrostatic interaction of the positively charged nascent peptide with the negatively charged ribosomal exit tunnel leads to translation arrest. When stalled long enough, the translation process is terminated with the degradation of the transcript and an incomplete protein. Although early experiments made a strong argument for this mechanism, other features associated with positively charged reporters, such as codon bias and mRNA and protein structure, have emerged as potent inducers of ribosome stalling. We carefully reviewed the published data on the protein and mRNA expression of artificial constructs with diverse compositions as assessed in different organisms. We concluded that, although polybasic sequences generally lead to lower translation efficiency, it appears that an aggravating factor, such as a nonoptimal codon composition, is necessary to cause translation termination events.

scholarly journals Ribosome profiling in Streptococcus pneumoniae reveals the role of methylation of 23S rRNA nucleotide G748 on ribosome stalling

2020 ◽  
Author(s):  
Tatsuma Shoji ◽  
Akiko Takaya ◽  
Yoko Kusuya ◽  
Hiroki Takahashi ◽  
Hiroto Kawashima
Keyword(s):  
Streptococcus Pneumoniae ◽  
Ribosome Profiling ◽  
23S Rrna ◽  
Ribosome Stalling ◽  
70S Ribosome ◽  
Exit Tunnel ◽  
Species Specific ◽  
Peptidyltransferase Center ◽  
First Time

2.Abstract(1) BackgroundMany nucleotides in 23S rRNA are methylated post-transcriptionally by methyltransferases and cluster around the peptidyltransferase center (PTC) and the nascent peptidyl exit tunnel (NPET) located in 50S subunit of 70S ribosome. Biochemical interactions between a nascent peptide and the tunnel may stall ribosome movement and affect expression levels of the protein. However, no studies have shown a role for NPET on ribosome stalling using an NPET mutant.(2) ResultsA ribosome profiling assay in Streptococcus pneumoniae demonstrates for the first time that an NPET mutant exhibits completely different ribosome occupancy compared to wild-type. We demonstrate, using RNA footprinting, that changes in ribosome occupancy correlate with changes in ribosome stalling. Further, statistical analysis shows that short peptide sequences that cause ribosome stalling are species-specific and evolutionarily selected. NPET structure is required to realize these specie-specific ribosome stalling.(3) ConclusionsResults support the role of NPET on ribosome stalling. NPET structure is required to realize the species-specific and evolutionary conserved ribosome stalling. These findings clarify the role of NPET structure on the translation process.

scholarly journals Arginine in C9ORF72 Dipolypeptides Mediates Promiscuous Proteome Binding and Multiple Modes of Toxicity

2020 ◽  
Vol 19 (4) ◽  
pp. 640-654 ◽  
Author(s):  
Mona Radwan ◽  
Ching-Seng Ang ◽  
Angelique R. Ormsby ◽  
Dezerae Cox ◽  
James C. Daly ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Arginine Methylation ◽  
Translation Elongation ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Exit Tunnel ◽  
Endogenous Proteins ◽  
Ribosome Exit Tunnel ◽  
Dipeptide Repeat

C9ORF72-associated Motor Neuron Disease patients feature abnormal expression of 5 dipeptide repeat (DPR) polymers. Here we used quantitative proteomics in a mouse neuronal-like cell line (Neuro2a) to demonstrate that the Arg residues in the most toxic DPRS, PR and GR, leads to a promiscuous binding to the proteome compared with a relative sparse binding of the more inert AP and GA. Notable targets included ribosomal proteins, translation initiation factors and translation elongation factors. PR and GR comprising more than 10 repeats appeared to robustly stall on ribosomes during translation suggesting Arg-rich peptide domains can electrostatically jam the ribosome exit tunnel during synthesis. Poly-GR also recruited arginine methylases, induced hypomethylation of endogenous proteins, and induced a profound destabilization of the actin cytoskeleton. Our findings point to arginine in GR and PR polymers as multivalent toxins to translation as well as arginine methylation that may explain the dysfunction of biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly.

scholarly journals Methylation of 23S rRNA G748 and the ribosomal protein L22 Lys-94 are critical factors for maintaining the association between ribosome stalling and proteome composition in Streptococcus pneumoniae

2021 ◽  
Author(s):  
Tatsuma Shoji
Keyword(s):  
Streptococcus Pneumoniae ◽  
Ribosomal Protein ◽  
Methylation Status ◽  
Ribosome Profiling ◽  
Whole Genome Sequence ◽  
23S Rrna ◽  
Gene Encoding ◽  
Ribosome Stalling ◽  
Exit Tunnel ◽  
High Level

AbstractBackground23S rRNA modification located at the nascent peptides exit tunnel plays an important role in both translation processes and the binding of the antibiotics. Methylation of the guanine at position 748 (m1G748) in 23S rRNA in Streptococcus pneumoniae is involved in the ribosome stalling and the binding of the antibiotic telithromycin (TEL). The disruption of the gene encoding RlmAII which methylates 23S rRNA G748 results in the increased resistance of TEL in S. pneumoniae. However, an isolated high-level TEL-resistant S. pneumoniae strain indicated that additional undescribed factors were involved in TEL resistance in S. pneumoniae.ResultsWe successfully isolated a high-level TEL-resistant S. pneumoniae RlmAII mutant and determined the whole-genome sequence. The lysine residue at the position 94 in ribosomal protein 22 (L22 K94) was critical in binding of TEL to the ribosome. A growth competition assay showed that L22 K94 was required for the function of m1G748. Ribosome profiling revealed that m1G748 and L22 K94 were both essential to maintain the relationship between the ribosome stalling and proteome composition.ConclusionIn S. pneumoniae, the combination of methylation status of G748 and the residue at position 94 in L22 are essential for both the distribution of ribosome stalling and the binding of TEL to ribosomes.

scholarly journals Electrostatic Interactions between Nascent Chains and the Ribosome Exit Tunnel Generate Forces that Modify Translation Rates

2021 ◽  
Vol 120 (3) ◽  
pp. 217a-218a
Author(s):  
Sarah E. Leininger ◽  
Judith Rodriguez ◽  
Quyen Vu ◽  
Yang Jiang ◽  
Mai S. Li ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

scholarly journals Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule

2018 ◽  
Author(s):  
Wenfei Li ◽  
Fred R. Ward ◽  
Kim F. McClure ◽  
Stacey Tsai-Lan Chang ◽  
Elizabeth Montabana ◽  
...  
Keyword(s):  
Small Molecules ◽  
Translation Termination ◽  
Structural Basis ◽  
Small Subset ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

AbstractSmall molecules that target the ribosome generally have a global impact on protein synthesis. However, the drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small subset of proteins by an unknown mechanism. In high-resolution cryo-electron microscopy (cryo-EM) structures of human ribosome nascent chain complexes stalled by PF846, PF846 binds in the ribosome exit tunnel in a newly-identified and eukaryotic-specific pocket formed by the 28S ribosomal RNA (rRNA), and redirects the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state that precedes mRNA and tRNA translocation, with peptidyl-tRNA occupying a mixture of A/A and hybrid A/P sites, in which the tRNA 3’-CCA end is improperly docked in the peptidyl transferase center. Using mRNA libraries, selections of PF846-dependent translation elongation stalling sequences reveal sequence preferences near the peptidyl transferase center, and uncover a newly-identified mechanism by which PF846 selectively blocks translation termination. These results illuminate how a small molecule selectively stalls the translation of the human ribosome, and provides a structural foundation for developing small molecules that inhibit the production of proteins of therapeutic interest.

scholarly journals Selective inhibition of human translation termination by a drug-like compound

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Wenfei Li ◽  
Stacey Tsai-Lan Chang ◽  
Fred. R. Ward ◽  
Jamie H. D. Cate
Keyword(s):  
Small Molecules ◽  
Translation Termination ◽  
Selective Inhibition ◽  
Helical Conformation ◽  
Eukaryotic Translation ◽  
Inhibit Gene Expression ◽  
Nascent Chain ◽  
Specific Proteins ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

Abstract Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.

scholarly journals The impact of ribosomal interference, codon usage, and exit tunnel interactions on translation elongation rate variation

2016 ◽  
Author(s):  
Khanh Dao Duc ◽  
Yun S. Song
Keyword(s):  
Electrostatic Interactions ◽  
Elongation Rate ◽  
Ribosome Profiling ◽  
Rate Variation ◽  
Biophysical Properties ◽  
Translation Elongation ◽  
Nascent Polypeptide ◽  
Exit Tunnel ◽  
Pass Through ◽  
The Impact

ABSTRACTPrevious studies have shown that translation elongation is regulated by multiple factors, but the observed heterogeneity remains only partially explained. To dissect quantitatively the different determinants of elongation speed, we use probabilistic modeling to estimate initiation and local elongation rates from ribosome profiling data. This model-based approach allows us to quantify the extent of interference between ribosomes on the same transcript. We show that neither interference nor the distribution of slow codons is sufficient to explain the observed heterogeneity. Instead, we find that electrostatic interactions between the ribosomal exit tunnel and specific parts of the nascent polypeptide govern the elongation rate variation as the polypeptide makes its initial pass through the tunnel. Once the N-terminus has escaped the tunnel, the hydropathy of the nascent polypeptide within the ribosome plays a major role in modulating the speed. We show that our results are consistent with the biophysical properties of the tunnel.

scholarly journals Selective inhibition of human translation by a drug-like compound that traps terminated protein nascent chains on the ribosome

2020 ◽  
Author(s):  
Wenfei Li ◽  
Stacey Tsai-Lan Chang ◽  
Fred. R Ward ◽  
Jamie H. D. Cate
Keyword(s):  
Small Molecules ◽  
Stop Codon ◽  
Translation Termination ◽  
Selective Inhibition ◽  
Helical Conformation ◽  
28S Rrna ◽  
Inhibit Gene Expression ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

AbstractMethods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation 1–4. These molecules also inhibit translation termination 4 by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 arrests translation at the stop codon by slowing hydrolysis of the protein nascent chain (NC) from peptidyl-site (P-site) tRNA by eukaryotic release factor 1 (eRF1). After NC hydrolysis from the P-site tRNA, PF846 traps the NC in the ribosome exit tunnel in a compact α-helical conformation that induces 28S rRNA nucleotide rearrangements propagating back to the ribosome peptidyl transferase center (PTC). Mutational analyses and human cell-based experiments elucidate the pivotal amino acids of the NC required for PF846-dependent termination arrest, all of which face the PF846 side of the ribosome exit tunnel. The structural and functional data support a model in which PF846 inhibits translation termination by inducing allosteric conformational rearrangements in the NC and PTC that suppress peptidyl-tRNA hydrolysis promoted by eRF1, and trap the NC in the ribosome exit tunnel. This unprecedented mechanism of action reveals new principles of translation termination and lays the foundation for new therapeutic strategies.

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