ABSTRACT
The proteostasis network relies on numerous feedback mechanisms to strike a balance between the rates of
protein synthesis and degradation, which is crucial for the maintenance of protein homeostasis. Proper tuning of
the rate of protein synthesis is also critical for the fidelity of cotranslational protein folding, which requires
coordination between the ribosome and various molecular chaperones. This translational regulation is especially
important for the fidelity of membrane protein (MP) biosynthesis, as the disruption of translational dynamics
appears to coincide with cotranslational misfolding and premature degradation. Nevertheless, it is currently
unclear how the translational machinery detects and responds to the cotranslational MP misfolding. In a recent
study of the topological properties of the Sindbis virus (SINV) structural polyprotein, our team found that the
translocon-mediated membrane integration of the nascent polypeptide stimulates ribosomal frameshifting and
the premature termination of translation. This work revealed that cotranslational (mis)folding can alter translation
through programmed ribosomal frameshifting (PRF), which is typically viewed as an RNA-mediated translational
recoding mechanism. In the following, we outline evidence suggesting translocon-mediated PRF occurs during
the translation of many human MPs, including several misfolding-prone MPs such as the cystic fibrosis
transmembrane conductance regulator (CFTR). We provide multiple lines of evidence that demonstrate that PRF
can occur at several “checkpoints” during CFTR synthesis, and show that a pathogenic mutation known to induce
cotranslational misfolding (¿F508) stimulates ribosomal frameshifting and the premature termination of CFTR
translation. Based on these findings, we hypothesize that PRF sites allow the ribosome to tune the processivity
of translation in response to conformational transitions in the nascent chain. To test this hypothesis, we will
assess how mutations and small molecules that alter cotranslational CFTR folding impacts the processivity of
translation at each PRF site. To gain structural insights into this ribosomal frameshifting mechanism, we will also
extend our studies on the SINV structural polyprotein. To map the sequence constraints of translocon-mediated
PRF, we measured the effects of 2,003 mutations on the efficiency of ribosomal frameshifting by deep mutational
scanning. Our preliminary results reveal several structural features that appear to be critical for PRF, including
a putative lipid-binding face within a nascent transmembrane domain and a helical segment within the ribosomal
exit tunnel. To determine how these structural features induce PRF, we propose a novel fusion of molecular
modeling, cellular biochemistry, and virology experiments to elucidate these structural features. Finally, we will
leverage these insights to develop sequence-based energetic predictions for the efficiency of PRF within integral
MPs. We will also characterize putative PRF sites in several disease-linked MPs in order to validate these
findings and explore the potential role of PRF in MP homeostasis. Together, these investigations will provide
fundamental insights into a novel cotranslational feedback mechanism and the molecular basis of disease.