Determining the Ribosome Binding Footprint in Escherichia Coli to Improve the Ribosome Binding Site Calculator Model of Translation Initiation

Open Access
Author:
Smith, Ashlee Elizabeth
Area of Honors:
Chemical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Howard M Salis, Thesis Supervisor
  • Themis Matsoukas, Honors Advisor
Keywords:
  • ribosome
  • binding
  • model
  • site
  • translation
  • initiation
  • engineering
  • RBS
  • calculator
Abstract:
In the last 20 years, the technology used to create synthetic DNA has greatly advanced. Today, DNA can now be synthesized 3 times as quickly and costs 5 times less. The availability of recombinant DNA systems provides flexibility for researchers in academia and industry trying to solve metabolic engineering problems and elucidate biological functions. One common metabolic engineering problem is developing a system to control the expression of a protein of interest over a range from very little to very large expression. Instead of generating large combinatorial libraries to find a sequence with a specific function, which is time consuming and inefficient, researchers are rationally altering the DNA sequence to achieve a desired attribute using information gained from biophysical modeling. One such alteration is the design of synthetic ribosome binding sites to improve the rate of translation initiation in order to increase the expression of protein. The Ribosome Binding Site Calculator (RBS) is software that utilizes a biophysical model of translation initiation to allow researchers to predict the translation initiation rate of a particular sequence or generate a synthetic RBS with a specified translation initiation rate. There are many interactions that take place during translation initiation. In order to improve the accuracy of the model’s predictions, one such interaction that we studied is the ribosomal footprint. The ribosome must unfold a section of messenger RNA (mRNA) after the start codon and load it into an mRNA channel. This channel can only accommodate single stranded mRNA. The number of nucleotides in the coding section the ribosome must unfold and load into the channel is the ribosomal footprint (FP). We hypothesized that a secondary structure contained in the ribosomal footprint must be unfolded and would greatly decrease protein expression, and that a secondary structure outside of the footprint region would not have to be unfolded and protein expression would be higher. We identified the ribosomal footprint for Escherichia coli by creating a high expressing RBS sequence and a modified coding section appended to the red fluorescent protein (RFP) reporter gene. The modified coding section featured a movable hairpin structure that was moved from immediately after ATG (position 3) to position 39 to show the changes in protein expression. We found that translation initiation gradually increased from very low levels when the hairpin was close to the start codon to over 1000-fold higher after the hairpin had passed the footprint region. The footprint was found to be 13 or 14 nucleotides including the start codon. Discrepancies between the model predictions and the data revealed the free energy the ribosome must expend to load the mRNA into the channel. The average error for constructs with a hairpin located 3 to 10 nucleotides into the coding sequence was determined to be 7.7 kcal/mol. This value represents the free energy cost of secondary structure within the footprint region, ΔGload. This information improves the biophysical model by determining the free energy change of loading the mRNA into the channel and will provide researchers with more accurate predictions of translation initiation.