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A Convenient Partnership: The Ribosome and the Nascent Chain Interact to Modulate Protein Synthesis and Folding

  • Author(s): Goldman, Daniel Hershel
  • Advisor(s): Bustamante, Carlos J
  • et al.
Abstract

During translation, the ribosome reads the genetic code of the messenger RNA, adding one amino acid at a time to the nascent protein. The sequence of the polypeptide determines the three dimensional structure of the natively folded protein, and thus encodes its biological activity. Because folding rates are often fast compared to translation, many proteins likely undergo folding transitions during synthesis, with folding potentially modulated by the sequential appearance of the polypeptide and the chemical environment of the ribosome. Traditional experimental approaches to study protein folding employ chemical, temperature or pH-induced denaturation and would irreversibly destroy the ribosome; thus, such techniques cannot be used to probe folding on the ribosome. In this work, we implement a novel single-molecule optical tweezers assay to probe folding transitions of the nascent polypeptide as it emerges from the ribosome. We demonstrate that the ribosome can modulate the kinetics of folding through interactions between the nascent chain and the charged ribosomal surface. Additionally, the ribosome can prevent misfolding of incompletely synthesized protein fragments. These observations point to a chaperone-like role for the ribosome in guiding the nascent protein to its native state.

In addition to interacting with the exterior of the ribosome, some nascent chain sequences can form specific contacts with the ribosome exit tunnel. These contacts lead to conformational changes of the ribosome, and reduced translation rates. The Secretion Monitor protein stalls the ribosome upon translation of a 17 amino acid motif. Arrest release requires targeting of the stalled ribosome-nascent chain complex to the translocon; thus, it has been hypothesized that arrest is released by a mechanical pulling force generated as the polypeptide is translocated across the membrane. By applying force to the nascent polypeptide of stalled ribosomes, we demonstrate that translation arrest at SecM is released by mechanical force. Additionally, we show that the force needed to release stalling can be generated by a protein folding in close proximity to the ribosome tunnel exit. Our results demonstrate the feasibility of a feedback mechanism, whereby a folding protein can modulate its synthesis through the generation of force. More generally, since the nascent polypeptide in the cell can undergo a number of potentially force-generating events–chaperone binding, protein or ligand binding, translocation and membrane insertion–force applied to the nascent chain may be an important modulator of protein synthesis.

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