RBS Calculator | protocol-book (2024)

RBS Calculator

Summary

The Ribosome Binding Site (RBS) Calculator is a design algorithm for predicting and controlling translation initiation and protein expression in bacteria across a 100,000-fold range. In Predict mode, the RBS Calculator calculates the translation initiation rate for every start codon in an mRNA transcript. In Design mode, the RBS Calculator generates an optimized synthetic RBS sequence to achieve a targeted translation initiation rate for an inputted protein coding sequence. Additional design constraints can be used to customize the synthetic RBS sequence, for example, to include restriction sites or a constant upstream sequence.

Web Interface Links

RBS Calculator Predict Mode

RBS Calculator Design Mode

RBS Calculator Predict Mode

Inputs

Title: the name of your design job [optional]

mRNA Sequence: the nucleotide sequence of the mRNA transcript, starting from the transcriptional start site and ending at the transcriptional terminator. ATCGU nucleotides allowed.

Host Organism: the bacterial species used to express the protein, selectable from a long list (start typing to narrow down your choices).

Free Energy Model Version: the free energy model used to carry out predictions. New versions are developed as more interactions are quantitatively characterized and incorporated into the model.

Outputs

Translation Initiation Rates: the calculated translation initiation rates for each start codon in the mRNA sequence. The rates are given on a proportional scale ranging from 1 to 100,000+.

Translated Open Reading Frames: a plot showing the translation initiation rates for each protein coding sequence in the mRNA transcript at their respective locations. mRNA transcripts often contain multiple start codons, yielding multiple (potentially overlapping) open reading frames. The open reading frames with the highest translation initiation rates are favored by the ribosome for synthesis of the corresponding proteins.

Ribosome Binding Free Energy Calculations: a detailed table showing the free energy calculations used to predict each start codon's translation initiation rate. Each Gibbs free energy change is the quantification of an interaction between the ribosome and mRNA that affects its translation initiation rate.

RBS Calculator Design Mode

Inputs

Title: the name of your design job [optional]

Protein Coding Sequence: the nucleotide sequence of the protein coding sequence, starting from the start codon and ending at the stop codon. ATCGU nucleotides allowed.

Target Translation Initiation Rate: the desired translation initiation rate for your protein coding sequence, given on a proportional scale from 1 to 100,000+. The maximum possible translation initiation rate is over 5,000,000; however, depending on the protein coding sequence, the maximum rate is not always achievable.

"Goal: Maximize Translation Initiation Rate": check this box to design a synthetic RBS sequence with the highest possible translation initiation rate for your protein coding sequence. Warning: an excessively high translation rate may become toxic to cells, due to ribosome sequestration and metabolic burden.

Host Organism: the bacterial species used to express the protein, selectable from a long list (start typing to narrow down your choices).

Advanced Options

Initial RBS Sequence: sequence used to initialize the design of the synthetic RBS (ATCGU nucleotides allowed). Useful when designing synthetic RBSs with very high translation initiation rates (e.g. entering a previously designed synthetic RBS as the starting point for a new design calculation). [optional]

Constant Upstream Sequence: a nucleotide sequence that appears upstream of the synthetic RBS (ATCGU nucleotides allowed). Useful when adding upstream restriction sites, ribozymes, or any other mRNA sequence (e.g. a long 5' UTR that should remain unchanged). The constant upstream sequence must be part of the mRNA transcript. [optional]

RBS Sequence Constraints: a degenerate nucleotide sequence that controls which nucleotides are allowed to be chosen during RBS design. Degenerate nucleotides follow the nucleotide IUPAC code (ATCGUSRMWKYVBDHN allowed). [optional]

Free Energy Model Version: the free energy model used to carry out predictions. New versions are developed as more interactions are quantitatively characterized and incorporated into the model. [optional]

Outputs

Designed RBS sequence: the ribosome binding site sequence generated by the RBS Calculator. The predicted translation initiation rate is also shown, specifically when this RBS sequence is placed upstream of the specified protein coding sequence.

Translated Open Reading Frames: a plot showing the translation initiation rates for each protein coding sequence in the mRNA transcript at their respective locations. Protein coding sequences often contain multiple start codons, yielding multiple (potentially overlapping) open reading frames. The open reading frames with the highest translation initiation rates are favored by the ribosome for synthesis of the corresponding proteins. It is important to examine this plot to ensure that the specified protein coding sequence does not have any internal start codons with higher translation initiation rates.

mRNA Stability Calculations: the predicted decay rate of the mRNA transcript, assuming that there is only one protein coding sequence in the operon. The calculations consider the sequence and structure of the 5' untranslated region as well as the translation rate of the protein coding sequence. The contributions to the prediction include RNase binding probabilities and the level of ribosome protection (quantified by the average unprotected distance between ribosomes).

Ribosome Binding Free Energy Calculations: a detailed table showing the free energy calculations used to predict each start codon's translation initiation rate. Each Gibbs free energy change is the quantification of an interaction between the ribosome and mRNA that affects its translation initiation rate. Read more about the RBS Calculator's free energy model.

RBS Calculator Free Energy Model

The RBS Calculator's free energy model quantifies the energetic (thermodynamic) interactions affecting the translation initiation rate of an individual start codon. These interactions are quantified in terms of Gibbs free energy changes, compared to a reference mRNA state in a system with constant temperature and pressure. The reference mRNA state is a fully unfolded mRNA that is not bound by the ribosome. Overall, the current equation for the RBS Calculator free energy model is:

$$\Delta G_{total} = \Delta G_{standby} + \Delta G_{mRNA-rRNA} +\Delta G_{spacing} + \Delta G_{start} + \Delta G_{stacking} - \Delta G_{mRNA}$$ΔGtotal​=ΔGstandby​+ΔGmRNA−rRNA​+ΔGspacing​+ΔGstart​+ΔGstacking​−ΔGmRNA​

From these calculations, the translation initiation rate $(r)$(r) of a start codon is then calculated according to Boltzmann's relationship:

$$r = exp(-\beta\Delta G_{total})$$r=exp(−βΔGtotal​)

The constant$\beta$βis a conversion factor from energy to probability under equilibrium conditions. In ideal (dilute) systems,$\beta=1/RT$β=1/RT where R is the gas constant and T is temperature. However, based on empirical measurements, the value for$\beta$β is about $0.45 \pm 0.05$0.45±0.05 mol/kcal inside the non-ideal (crowded) environment inside the cell.

The major interactions controlling translation initiation rate are:

1. Initial binding of the 30S small ribosomal subunit to upstream standby sites. Upstream standby sites with different geometric accessibilities and structural unfolding free energies will lead to different rates of initial ribosome binding. The Gibbs free energy term $\Delta G_{standby}$ΔGstandby​ quantifies how well the 30S ribosomal subunit can bind to upstream standby sites. It is either zero for a fully accessible site and becomes more positive as the site is less favorably bound. There are three components to this free energy term: $\Delta G_{distortion}$ΔGdistortion​, $\Delta G_{unfolding}$ΔGunfolding​, and $\Delta G_{sliding}$ΔGsliding​.

2. Unfolding of mRNA structures that overlap with the ribosomal footprint surrounding the start codon. mRNAs fold into structures. Before a ribosome can initiate translation, it must unfold any mRNA structures that overlap with its binding site. The ribosome's binding site (its physical footprint) extends from the 5' end of the 16S rRNA binding site to 13 nucleotides after the start codon. The Gibbs free energy term $-\Delta G_{mRNA}$−ΔGmRNA​ is the energy needed to unfold all mRNA structures within this region. More stable mRNA structures require more energy to unfold, leading to lower translation initiation rates.

3. Hybridization between mRNA and the 16S rRNA. The superstructure of the 30S ribosomal subunit is made up of the 16S rRNA. The last 9 nucleotides of the 16S rRNA are accessible (in most bacteria) and is used by the ribosome to stabilize binding to mRNAs. Hybridization between the mRNA and 16S rRNA occurs at a sequence commonly known as the Shine-Dalgarno. The Gibbs free energy term $\Delta G_{mRNA-rRNA}$ΔGmRNA−rRNA​ is the quantification of this hybridization energy together with other mRNA-mRNA interactions do not require unfolding during translation initiation. A more negative free energy indicates a more favorable interaction at the Shine-Dalgarno sequence.

4. Start codon-tRNA interactions. Inside the ribosome, the start codon base pairs to the tRNA-fMet. The canonical start codon AUG has perfect complimentary to the anti-codon loop of tRNA-fMet and therefore has the highest binding free energy. However, other non-canonical start codons (GUG, CUG, and UUG) can still base pair to tRNA-fMet, but with reduced binding free energies. The Gibbs free energy term $\Delta G_{start}$ΔGstart​ is the free energy released when the start codon base pairs to tRNA-fMet. Translation initiation factors (IFs) can additionally alter these binding free energies.

5. Stretching or compression of the ribosome by the spacer region. The length of mRNA between the 16S rRNA binding site (Shine-Dalgarno sequence) and the start codon is called the spacer region. There is an optimal spacer length where the ribosome forms both contacts with distortion. However, if the spacer region is too long or too short, it causes the ribosome to stretch or compress, resulting in an energetic penalty. The Gibbs free energy term $\Delta G_{spacing}$ΔGspacing​ is the free energy penalty for stretching or compression of the 30S ribosomal subunit when bound. This term is zero when the spacer length is optimal and positive when the ribosome is either stretched or compressed.

6. Certain motifs form unusual mRNA structures that alter ribosome binding. Long stretches of hom*opolymer sequence in the spacer region cause stacking interactions (quantified by $\Delta G_{stacking}$ΔGstacking​). G-rich sequences may form unusually stable G-quadruplex structures. Pseudoknots may also form between long distance nucleotides that alter structure formation.

7. Ribosome Drafting is a non-equilibrium phenomenon that increases a mRNA's translation initiation rate. Ribosome Drafting takes place when a mRNA with slow-folding structures is successively bound by fast-binding ribosomes. Here, the mRNA structures do not have enough time to refold, eliminating the energetic penalty for unfolding these structures. The presence of Ribosome Drafting adds a kinetic component to determining the free energy needed to unfold mRNA structures,$\Delta G_{mRNA}$ΔGmRNA​.

8. Certain motifs bind RNA-binding proteins that alter ribosome binding. Examples include CsrA and Hfq in Escherichia coli.

Relevant Articles

Reis, A.C. & Salis, H.M. (2020). An automated model test system for systematic development and improvement of gene expression models. ACS Synthetic Biology, 9(11), 3145-3156.

Cetnar, D. P., & Salis, H. M. (2020). Systematic Quantification of Sequence and Structural Determinants Controlling mRNA stability in Bacterial Operons. bioRxiv.

Espah Borujeni, A., Cetnar, D., Farasat, I., Smith, A., Lundgren, N., & Salis, H. M. (2017). Precise quantification of translation inhibition by mRNA structures that overlap with the ribosomal footprint in N-terminal coding sequences. Nucleic acids research, 45(9), 5437-5448.

Espah Borujeni, A. & Salis, H.M. (2016). Translation initiation is controlled by RNA folding kinetics via a ribosome drafting mechanism. Journal of the American Chemical Society, 138(22), 7016-7023.

Espah Borujeni, A., Channarasappa, A.S., & Salis, H.M. (2013). Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic acids research, 42(4), 2646-2659.

Salis, H.M., Mirsky, E.A., & Voigt, C.A. (2009). Automated design of synthetic ribosome binding sites to control protein expression. Nature biotechnology, 27(10), 946.