Synonymous codons affect polysome spacing, protein production and protein folding stress: studies of bacterial translation using ribosome profiling

ribosome;translation rate;co-translational protein folding;synonymous codons;firefly luciferase;molecular chaperones;heat shock response;Biology

Martens, Andrew TedSchleif, Robert F ;

Johns Hopkins University

关键词: ribosome;    translation rate;    co-translational protein folding;    synonymous codons;    firefly luciferase;    molecular chaperones;    heat shock response;    Biology;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60203/MARTENS-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The acquisition of protein secondary and tertiary structure depends on the primary sequence of amino acids. However, predicting a protein;;s folded structure is difficult even with the knowledge of its sequence. It has been suggested that, in addition to encoding the amino acid sequence, genes also encode kinetic information which regulates the ribosome;;s translation rate. This information might guide nascent protein folding during translation.With the advent of ribosome profiling, a high-throughput sequencing technique which quantifies ribosome density on mRNA, it is now possible to investigate this hypothesis in greater detail. Here, a new way to analyze ribosome profiling data is presented, confirming that ribosome profiling detects ribosome pauses at slow codons. This method is able to precisely determine the locations of the ribosome aminoacyl and peptidyl transfer sites within the ribosome footprint. Next, a simulation tool which models the progression of ribosomes along an mRNA is used to explore the effects of translation initiation and elongation rates on protein expression. This tool can be used to generate testable predictions for how changing the translation rate should affect various experimental observables, including ribosome density. New experimental data, collected from the bacterium Escherichia coli, demonstrate that the sequence of the Firefly (Photinus pyralis) Luciferase mRNA affects its ribosome occupancy. Importantly, ribosome occupancy is differentially influenced by synonymous codons.These data also show that Luc expression is controlled by the 15 codons immediately downstream of the start codon and that greater Luciferase expression levels progressively activate the heat shock response. However, this response appears to saturate, suggesting that the overexpression of foreign proteins in E. coli readily overwhelms the endogenous chaperone system. This result demonstrates that expression level, rather than translation kinetics, determines the yield of folded Luciferase protein in E. coli.