PT  - JOURNAL ARTICLE
AU  - Amin Espah Borujeni
AU  - Daniel Cetnar
AU  - Iman Farasat
AU  - Ashlee Smith
AU  - Natasha Lundgren
AU  - Howard M Salis
TI  - Precise Quantification of Translation Inhibition by mRNA Structures that Overlap with the Ribosomal Footprint in N-terminal Coding Sequences
AID  - 10.1101/091470
DP  - 2016 Jan 01
TA  - bioRxiv
PG  - 091470
4099  - http://biorxiv.org/content/early/2016/12/04/091470.short
4100  - http://biorxiv.org/content/early/2016/12/04/091470.full
AB  - A mRNA’s translation rate is controlled by several sequence determinants, including the presence of RNA structures within the N-terminal regions of its coding sequences. However, the physical rules that govern when such mRNA structures will inhibit translation remain unclear. Here, we introduced systematically designed RNA hairpins into the N-terminal coding region of a reporter protein with steadily increasing distances from the start codon, followed by characterization of their mRNA and expression levels in E. coli. We found that the mRNAs’; translation rates were repressed, by up to 1410-fold, when mRNA structures overlapped with the ribosome’s footprint. In contrast, when the mRNA structure was located outside the ribosome’s footprint, translation was repressed by less than 2-fold. By combining our measurements with biophysical modeling, we determined that the ribosomal footprint extends 13 nucleotides into the N-terminal coding region and, when a mRNA structure overlaps or partially overlaps with the ribosomal footprint, the free energy to unfold only the overlapping structure controlled the extent of translation repression. Overall, our results provide precise quantification of the rules governing translation initiation at N-terminal coding regions, improving the predictive design of post-transcriptional regulatory elements that regulate translation rate.