Impetuses for Rational Gene Design

Oliver Hendy

Degeneracy in the genetic code allows for a single protein
to be encoded for by many synonymous mRNA
transcripts. Silent mutations, while preserving the
integrity of the amino acid sequence, may affect expression
of the gene at the ribosome. Several factors, including
codon bias, mRNA secondary structures, and codon
context result in upper limits of protein production. Most
studies have used random silent mutations to study the
effects of these various factors. Rational gene design and
synthesis provides a foundation for controlled experiments
that can investigate the

Codon Bias
Codon bias, the non-random distribution of synonymous
codon usage, varies from species to species, and is thought to
be a result of cytoplasmic tRNA concentrations, where relative
codon requency corresponds to relative tRNA frequency
(Novoa & Ribas de Pouplana 2012). Optimizing genes for
expression by replacing low-frequency codons with their
preferred high-frequency codons has been shown to increase
protein production (References). Codon Adaptation Index
(CAI), a measure of overall codon fitness in a given mRNA
transcript relative to a highly expressed gene set, has been
used to explain and predict variation in gene expression
among homologous transcripts (Welch et. al. 2009). Codons
Bias has also been used to reduce genome efficiency in viruses
for attenuation (Mueller et. al. 2006).

It is debated whether this is a result of faster elongation or
greater translational accuracy ( , Qian et. al. 2012).

mRNA Structure
Secondary mRNA structure in the 5 untranslated region and
near the start codon is known to inhibit ribosome loading and
translation initiation. A number of randomly mutated GFP
transcripts were analyzed by Kudla et. al., and they explained
variation in expression with mRNA secondary structure in the
first 47 nucleotides of the transcript (2009). Borujeni et. al.
provide direct evidence for this hypothesis by showing that
secondary structure inhibits mRNA loading onto the 30s
subunit and that partial unwinding of this mRNA usually
occurs before accommodation by the ribosome (2013).

While Kudla. et. al. 2009 assert that mRNA 2 structure is the
main regulator of translation speed by controlling translation
initiation, Welch et. al. show that codon bias is a good
predictor of translational output. Certain datapoints in Welchs
paper could point to mRNA structures being a limiting factor,
however Qian. et. al. provide evidence that preferred codons
do not

Codon Context
There is growing evidence that tRNA-anticodon interactions
are not independent events, but influenced by their
environment in the mRNA transcript.
Codon Pair Bias
Codon pair bias, similar to codon bias, is the use of preferred
codon pairs. (Chevance et. al. 2014) Codon Pair Bias has been
used in a similar manner as codon bias to attenuate viruses
(Coleman et. al. 2008).
Codon Reuse
Codon reuse or autocorrelation, is the grouping of
synonymous codons in distinct regions of the mRNA
transcript. Codon reuse is thought to be a result of tRNA
recycling, where a single tRNA is used, recharged and used
again for the same transcript (Godinic-Mikulcic et. al. 2014).

Shine-Dalgarno Sequences


References

Borujeni, A. E., 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, gkt1139.

Cannarozzi, G., Schraudolph, N. N., Faty, M., von Rohr, P., Friberg,
M. T., Roth, A. C., ... & Barral, Y. (2010). A role for codon order in
translation dynamics. Cell, 141(2), 355-367.

Chevance, F. F., Le Guyon, S., & Hughes, K. T. (2014). The Effects
of Codon Context on In Vivo Translation Speed. PLoS genetics,
10(6), e1004392.

Coleman, J. R., Papamichail, D., Skiena, S., Futcher, B., Wimmer, E.,
& Mueller, S. (2008). Virus attenuation by genome-scale changes in
codon pair bias. Science, 320(5884), 1784-1787.

Godinic-Mikulcic, Vlatka, et al. "Archaeal aminoacyl-tRNA
synthetases interact with the ribosome to recycle tRNAs." Nucleic
acids research (2014): gku164.

Gu, W., Zhou, T., & Wilke, C. O. (2010). A universal trend of
reduced mRNA stability near the translation-initiation site in
prokaryotes and eukaryotes. PLoS computational biology, 6(2).

Kudla, G., Murray, A. W., Tollervey, D., & Plotkin, J. B. (2009).
Coding-sequence determinants of gene expression in Escherichia
coli. science, 324(5924), 255-258.

Li, G. W., Oh, E., & Weissman, J. S. (2012). The anti-Shine-
Dalgarno sequence drives translational pausing and codon choice in
bacteria. Nature, 484(7395), 538-541.

Mueller, S., Papamichail, D., Coleman, J. R., Skiena, S., & Wimmer,
E. (2006). Reduction of the rate of poliovirus protein synthesis
through large-scale codon deoptimization causes attenuation of viral
virulence by lowering specific infectivity. Journal of
virology, 80(19), 9687-9696.

Novoa, E. M., & Ribas de Pouplana, L. (2012). Speeding with
control: codon usage, tRNAs, and ribosomes. Trends in Genetics,
28(11), 574-581.

Qian, W., Yang, J. R., Pearson, N. M., Maclean, C., & Zhang, J.
(2012). Balanced codon usage optimizes eukaryotic translational
efficiency. PLoS genetics, 8(3), e1002603.

Welch, M., Govindarajan, S., Ness, J. E., Villalobos, A., Gurney, A.,
Minshull, J., & Gustafsson, C. (2009). Design parameters to control
synthetic gene expression in Escherichia coli. PloS one, 4(9), e7002.