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      Cotranslational protein folding on the ribosome monitored in real time.

      Science (New York, N.Y.)
      American Association for the Advancement of Science (AAAS)

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          Abstract

          Protein domains can fold into stable tertiary structures while they are synthesized on the ribosome. We used a high-performance, reconstituted in vitro translation system to investigate the folding of a small five-helix protein domain-the N-terminal domain of Escherichia coli N5-glutamine methyltransferase HemK-in real time. Our observations show that cotranslational folding of the protein, which folds autonomously and rapidly in solution, proceeds through a compact, non-native conformation that forms within the peptide tunnel of the ribosome. The compact state rearranges into a native-like structure immediately after the full domain sequence has emerged from the ribosome. Both folding transitions are rate-limited by translation, allowing for quasi-equilibrium sampling of the conformational space restricted by the ribosome. Cotranslational folding may be typical of small, intrinsically rapidly folding protein domains.

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          Most cited references16

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          EF-P is essential for rapid synthesis of proteins containing consecutive proline residues.

          Elongation factor P (EF-P) is a translation factor of unknown function that has been implicated in a great variety of cellular processes. Here, we show that EF-P prevents ribosome from stalling during synthesis of proteins containing consecutive prolines, such as PPG, PPP, or longer proline strings, in natural and engineered model proteins. EF-P promotes peptide-bond formation and stabilizes the peptidyl-transfer RNA in the catalytic center of the ribosome. EF-P is posttranslationally modified by a hydroxylated β-lysine attached to a lysine residue. The modification enhances the catalytic proficiency of the factor mainly by increasing its affinity to the ribosome. We propose that EF-P and its eukaryotic homolog, eIF5A, are essential for the synthesis of a subset of proteins containing proline stretches in all cells.
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            A pause for thought along the co-translational folding pathway.

            A unifying concept that combines the basic features governing self-organization of proteins into complex three-dimensional structures in vitro and in vivo is still lacking. Recent experimental results and theoretical in silico modeling studies provide evidence showing that mRNA might contain an additional layer of information, beyond the amino acid sequence, that fine-tunes in vivo protein folding, which is largely believed to start as a co-translational process. These findings indicate that translation kinetics might direct the co-translational folding pathway and that translational pausing at rare codons might provide a time delay to enable independent and sequential folding of the defined portions of the nascent polypeptide emerging from the ribosome.
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              Fluorescence quenching by photoinduced electron transfer: a reporter for conformational dynamics of macromolecules.

              Photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties, for example, the amino acid tryptophan or the nucleobase guanine, can quench fluorescence upon van der Waals contact and thus report on molecular contact. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and oligonucleotides. Whereas dynamic quenching transiently influences quantum yield and fluorescence lifetime of the fluorophore, static quenching in pi-stacked complexes efficiently suppresses fluorescence emission over time scales longer than the fluorescence lifetime. Static quenching therefore provides sufficient contrast to be observed at the single-molecule level. Here, we review complex formation and static quenching of different fluorophores by various molecular compounds, discuss applications as reporter system for macromolecular dynamics, and give illustrating examples.
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                Author and article information

                Journal
                26612953
                10.1126/science.aad0344

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