Effects of Translation Rate Changes on Protein Production, Folding and Fragmentation

In the cell, proteins are synthesized from N- to C-terminus by the ribosome. Once emerged from the ribosome exit tunnel, the nascent chain can begin to fold while its C-terminus is still untranslated or constrained inside the ribosome. The conformations of nascent chains once they emerge from the exit tunnel, but before an entire structural domain has appeared, are poorly understood but are known to affect folding pathways and yield. Translation rate can be slower than the formation of stable secondary and tertiary structure, meaning that altering translation rate could potentially influence the folding pathway and even the final, folded structure of a protein. Synonymous codon substitutions, which are known to alter local elongation rates, have been shown to affect the final structure of certain proteins. However, the effects of non-synonymous mutations have not been explored. In this thesis, I show that non-synonymous mutations known to affect translation rate can predictably alter the folded state of a protein biosensor. Additionally, a novel translation stall sequence composed of amino acids and stop codon usage components is characterized.

In addition to its effects on protein folding, stalling of translation elongation can also lead to the degradation of nascent polypeptides in both prokaryotes and eukaryotes. Protease degradation of these stalled nascent polypeptides requires addition of a peptide tag by quality control machinery. I demonstrate that the fragmentation of a model protein is controlled by both its translation rate and its C-terminal sequence polarity.

The conformations nascent polypeptide chains adopt while still tethered to the ribosome are not well-characterized. The conformations of proteins composed of primarily β-sheet structure, which contains many long-range interactions, are particularly unknown. Here, I describe the development of a technique to probe the surface exposure of a model β-sheet protein (GFP) on the ribosome using covalent labeling of ribosome-GFP-nascent chain complexes coupled to mass spectrometry. A method to isolate ribosome-nascent chain complexes from released GFP was presented and the association of nascent GFP with the ribosome in the complexes was verified.