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Electrostatic Interactions Govern Extreme Nascent Protein Ejection Times from Ribosomes and Can Delay Ribosome Recycling.

ABSTRACT: The ejection of nascent proteins out of the ribosome exit tunnel, after their covalent bond to transfer-RNA has been broken, has not been experimentally studied due to challenges in sample preparation. Here, we investigate this process using a combination of multiscale modeling, ribosome profiling, and gene ontology analyses. Simulating the ejection of a representative set of 122 E. coli proteins we find a greater than 1000-fold variation in ejection times. Nascent proteins enriched in negatively charged residues near their C-terminus eject the fastest, while nascent chains enriched in positively charged residues tend to eject much more slowly. More work is required to pull slowly ejecting proteins out of the exit tunnel than quickly ejecting proteins, according to all-atom simulations. An energetic decomposition reveals, for slowly ejecting proteins, that this is due to the strong attractive electrostatic interactions between the nascent chain and the negatively charged ribosomal-RNA lining the exit tunnel, and for quickly ejecting proteins, it is due to their repulsive electrostatic interactions with the exit tunnel. Ribosome profiling data from E. coli reveals that the presence of slowly ejecting sequences correlates with ribosomes spending more time at stop codons, indicating that the ejection process might delay ribosome recycling. Proteins that have the highest positive charge density at their C-terminus are overwhelmingly ribosomal proteins, suggesting the possibility that this sequence feature may aid in the cotranslational assembly of ribosomes by delaying the release of nascent ribosomal proteins into the cytosol. Thus, nascent chain ejection times from the ribosome can vary greatly between proteins due to differential electrostatic interactions, can influence ribosome recycling, and could be particularly relevant to the synthesis and cotranslational behavior of some proteins.

SUBMITTER: Nissley DA

PROVIDER: S-EPMC7312765 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mutations that cause Huntington's Disease involve a polyglutamine (polyQ) sequence expansion beyond 35 repeats in exon 1 of Huntingtin. Intracellular inclusion bodies of mutant Huntingtin protein are a key feature of Huntington's disease brain pathology. We previously showed that in cell culture the formation of inclusions involved the assembly of disordered structures of mHtt exon 1 fragments (Httex1) and they were enriched with translational machinery when first formed. We hypothesized that nascent mutant Httex1 chains co-aggregate during translation by phase separation into liquid-like disordered aggregates and then convert to more rigid, amyloid structures. Here we further examined the mechanisms of inclusion assembly in a human epithelial kidney (AD293) cell culture model. We found mHttex1 did not appear to stall translation of its own nascent chain, or at best was marginal. We also found the inclusions appeared to recruit low levels of RNA but there was no difference in enrichment between early formed and mature inclusions. Proteins involved in translation or ribosome quality control were co-recruited to the inclusions (Ltn1 Rack1) compared to a protein not anticipated to be involved (NACAD), but there was no major specificity of enrichment in the early formed inclusions compared to mature inclusions. Furthermore, we observed co-aggregation with other proteins previously identified in inclusions, including Upf1 and chaperone-like proteins Sgta and Hspb1, which also suppressed aggregation at high co-expression levels. The newly formed inclusions also contained immobile mHttex1 molecules which points to the disordered aggregates being mechanically rigid prior to amyloid formation. Collectively our findings show little evidence that inclusion assembly arises by a discrete clustering of stalled nascent chains and associated quality control machinery. Instead, the machinery appear to be recruited continuously, or secondarily, to the nucleation of inclusion formation.

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Electrostatic Interactions Govern Extreme Nascent Protein Ejection Times from Ribosomes and Can Delay Ribosome Recycling.

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