dG vs availability of RBS

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skadbio
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Joined: 08/19/2011

May I know, in gene expression studies:
1. which criteria is more important to produce the protein as high as possible:
dG of the mRNA secondary structure or the availability of ribosome binding site (ATG and kozak sequence, ...)
changing the codons according to the codon bias of the host results in lower dG (less negative dG) but available RBS, while considering the sequence without codon optimization makes the ATG and kozak sequence to be restricted but with higher dG.
2. How many bases should be considered in mfold when the size of gene is big.

zukerm
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Joined: 11/12/2010
Translation efficiency

You ask profound questions to which no one has a complete answer. I have some comments. I hope they will be useful to you and others. The ΔG of the mRNA should not be important. In fact, I am of the opinion that it is generally a waste of time to simulate foldings of mRNAs. If you have an mRNA that does not express, then it is probably worthwhile to compute secondary structures. You might find strong secondary structures that block ribosome binding. If the gene is large, I would fold the 5'-UTR and perhaps 100 nts into the coding region. You could make synonymous base changes to disrupt putative secondary structures.

There are codon and di-codon preferences for each host. It is not sufficient to use host preferred codons. You must also use di-codon preferences. However, if you optimize for host specific codon and di-codon preferences, you might have high expression, but the product might not fold into an active protein. There is an art to using some rare codons or non-preferred di-codons to force pauses in the translation, thereby facilitating the correct folding of the protein. Has anyone actually proved this? I would say no. What is true is that some proteins could be expressed in a host but did not fold properly. In many cases, the expressed protein formed inclusion bodies. In these cases, altering codon and di-codon usage has resulted in synthetic genes that express well. This technology was developed by CODA Genomics, now named Verdezyne.