Annotated Bib

mRNA secondary structure

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.

Looked at codon bias in several versions of GFP in E. Coli. Found no significant correlation between
CAI and fluorescene, suggesting that codon adaptation was not the rate limiting step in endogenous
expression. However, when a UTR was inserted upstream of AUG that had little/no secondary mRNA
structure, expression was uniformly increased. This suggests that past a certain point, increasing
codon optimization reaches diminishing returns if 5' UTR secondary structure is not similarly
optimized. Kudla et. al. Further hypothesize that CAI is not a cause of high expression, but rather that
high expression causes optimized CAI. This was confusing to me, but the premise of the argument as I
understand it is that translation initiation is sole regulator of post transcriptional regulation in the
ribosome, and that CAI either serves to reduce cell energy costs (by fewer sequestered ribosomes or
fewer misfolded proteins)



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.

Extremely detailed paper looking into secondary strucutre in the 5' UTR and how this affects gene
expression. Major findings include that secondary structure inhibits mRNA loading onto the 30s
subunit while surrounding linear mRNA promotes this loading. Unwinding of this secondary structure
can improve ribosome loading but requires input of free energy. Moreover, standby site regulates
expression independently of UTR length. Finally, the standyby site represents a significant target for
gene regulation via cis or trans acting factors (stem-loop structures, miRNA, etc.). Key takeaway:
Secondary structure in 5' UTR is a primary factor in determining level of gene expression by
controlling translation initiation.

Question: Is secondary structure REQUIRED for a standby structure to be able to load onto a
ribosome?



Need to find: In one paper, they assert that secondary structure in the coding region is less important to
avoid because ribosome has GTP powering it so it just trucks through them.


Codon Bias


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.

Qian et al take a cell wide sample of nucleotides associated with ribosome. The assumption made is
that the time it takes for a tRNA to find its codon is inversely proportional to the frequency of that
codon in the ribosome. For example, a codon that takes a long time to associate with its respective
tRNA should be found associated in the ribosome site more frequently than one that finds it quickly.

The results show that there is no significant difference in the frequencies of biased codons in the
binding site, suggesting that these codon biases do not promote faster translation. This further supports
the other generally accepted position that codon bias is selected for due to more accurate translation.

Questions:
How is this reconciled with tRNA concentration. Shouldn't tRNA's that have a higher
cytoplasmic concentration locate their respective codon more quickly?

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.

(Need to read more closely and write more)

This paper shows a rational design approach to gene design, and investigates the role of synonymous
codon bias on expression of DNA Pol and Single chain antibody in E. coli. Contradictary to Plotikin
(2009), Welch and colleagues provide evidence that synonymous codon bias is the result of variation in
expression of various forms of their genes.


However, a compromise is found in some of the data, where a highly deleterious 5' region was found
to drastically decrease expression despite good codon bias. This suggests that both translation
initiaion and elongation can be rate limiting, and depending on the sequence, either can be the
limiting step in gene expression. In Plot

Question: Could it be possible to analyze sequences from Plotkins paper vs Welch paper and determine
if there were different limiting steps in the two studies that lead to the two different conclusions.


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.


Liu, B., & Qian, S. B. (2014). Translational reprogramming in cellular stress response. Wiley
Interdisciplinary Reviews: RNA, 5(3), 301-305.

Describes mechanisms by which translation can be altered under stress conditions. Review describes
how in one study it was found that the tRNA concentration of a methylated leucine tRNA increased.

Other interesting papers

Wald, N., Alroy, M., Botzman, M., & Margalit, H. (2012). Codon usage bias in prokaryotic
pyrimidine-ending codons is associated with the degeneracy of the encoded amino acids. Nucleic
acids research, 40(15), 7074-7083.

Chan CT, Pang YL, Deng W, Babu IR, Dyavaiah
M, Begley TJ, Dedon PC. Reprogramming of tRNA
modifications controls the oxidative stress response by
codon-biased translation of proteins.
Nat Commun
2012, 3:937.

Codon Context


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.

This study investigates the role of codon order in gene expression. They hypothesize that repetition of
specific codons that have synonymous counterparts for a single amino acid increases translation speed.
Using several synthesized versions of GFP, they do find that codon reuse results in significantly
improved gene expression.

There are several possible explanations for this phenomenon, but the most plausible can be explained
by the fact that ribosome elongation is significantly more rapid than the rate of tRNA diffusion away
from the ribosome. In addition, tRNAs are recharged

Questions: Is this more important than codon bias? Should optimizer programs try to resolve this
problem and sacrifice codon bias or vice versa?


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

Awesome review article that looks at the many determinants of gene expression. Not just about codon
context, but has some important takeaways. Expression of different gene sets can be regulated by
tRNA modifciations. This is of significance to us because choosing a reference set must be done
appropriately to reflect the actual experimental conditions. Preferred codons is not an absolute term,
since this can change due to environmental factors, and more directly, tRNA modifications. Therefore,
a codon bias should be defined with reference to the conditions (Highly expressed in optimal
conditions, highly expressed in stress conditions, etc.).






Codon Usage and Selection on Proteins