Computational protocol: Efficient search, mapping, and optimization of multi protein genetic systems in diverse bacteria

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Protocol publication

[…] The RBS Calculator v1.1 was employed to calculate the ribosome's binding free energy to bacterial mRNA sequences, and to predict the translation initiation rate of a protein-coding sequence on a proportional scale that ranges from 0.1 to 100,000 or more. The thermodynamic model uses a 5-term Gibbs free energy model to quantify the strengths of the molecular interactions between the 30S ribosomal pre-initiation complex and the mRNA region surrounding a start codon. The free energy model is: (1) Using statistical thermodynamics and assuming chemical equilibrium between the pool of free 30S ribosomes and mRNAs inside the cell, the total Gibbs free energy change is related to a protein-coding sequence's translation initiation rate, r, according to: (2) This relationship has been previously validated on 132 mRNA sequences where the ΔGtotal varied from −10 to 16 kcal/mol, resulting in well-predicted translation rates that varied by over 100,000-fold (Salis et al, ). The apparent Boltzmann constant, β, has been measured as 0.45 ± 0.05 mol/kcal, which was confirmed in a second study (Hao et al, ). In practice, we use a proportional constant of 2,500 to generate a proportional scale where physiological common translation initiation rates vary between 1 and 100,000 au.In the initial state, the mRNA exists in a structured conformation, where its free energy of folding is ΔGmRNA (ΔGmRNA is negative). After assembly of the 30S ribosomal subunit, the last nine nucleotides of its 16S rRNA have hybridized to the mRNA while all non-clashing mRNA structures are allowed to fold. The free energy of folding for this mRNA–rRNA complex is ΔGmRNA:rRNA (ΔGmRNA:rRNA is negative). mRNA structures that impede 16S rRNA hybridization or overlap with the ribosome footprint remain unfolded in the final state. These Gibbs free energies are calculated using a semi-empirical free energy model of RNA and RNA–RNA interactions (Xia et al, ; Mathews et al, ) and the minimization algorithms available in the Vienna RNA suite, version 1.8.5 (Gruber et al, ).Three additional interactions will alter the translation initiation rate. The tRNAfMET anti-codon loop hybridizes to the start codon (ΔGstart is most negative for AUG and GUG). The 30S ribosomal subunit prefers a five-nucleotide distance between the 16S rRNA-binding site and the start codon; non-optimal distances cause conformational distortion and lead to an energetic binding penalty. This relationship between the ribosome's distortion penalty (ΔGspacing > 0) and nucleotide distance was systematically measured. Finally, the 5′ UTR binds to the ribosomal platform with a free energy penalty ΔGstandby.There are key differences between the first version of the RBS Calculator (v1.0) (Salis et al, ) and version v1.1 (Salis, ). The algorithm's use of free energy minimization was modified to more accurately determine the 16S rRNA-binding site and its aligned spacing, particularly on mRNAs with non-canonical Shine-Dalgarno sequences, and to accurately determine the unfolding free energies of mRNA structures located within a protein-coding sequence. For the purpose of this work, a ribosome-binding site (RBS) sequence is defined as the 35 nucleotides located before the start codon of a protein-coding sequence within a mRNA transcript. However, the presence of long, highly structured 5′ UTRs can further alter the translation initiation rate of a protein-coding sequence by manipulating its ΔGstandby. The ribosome's rules for binding to long, highly structured 5′ UTRs has been characterized (Espah Borujeni et al, 2014) and will be incorporated into a future version of the RBS Calculator (v2.0). […]

Pipeline specifications

Software tools RBS Calculator, ViennaRNA
Application Synthetic biology