Motivation: understand codon bias.
Hyp. 1: using common codons will allow higher expression
- Sub: CAI in only part of gene?
Hyp. 2: codon usage will affect RNA folding and expression levels
- Sub: Only in part of gene?
Fig. 1: create lots of pieces of gene with degenerate nucleotides. These are nucleotides that can pair with multiple other nucleotides
PCR amplify these: what will happen ? Will get randomized nucleotides at 3rd position
Put these into vector, with T7 promoter which is very specific for T7 polymerase (thus high expression). Insert into bacteria with T7 polymerase -> high expression.
F1 is origin of replication
MCS is a site engineered with lots of restriction enzyme (what is? )sites so you can insert sequence easily.
What's the amp ? To know your transformation worked.
Why the lacI ? So you can induce transcription by putting bacteria on medium with low lactose and high galactose!
How to calculate CAI: show math. baseline is comparison of highly expressed gene set (a bit circular!)
Fig1: their genes representative of range seen in E. coli.
High CAI means ? codon w/ lots of tRNA.
What's a biological replicate ? vs. technical. (weighing tiger vs. great white sharks example) So measuring same insertion event multiple times/ways -> technical. Repeating insertion event -> biological replicate.
Why test alternate bacterial promoter ? Rule out some T7 specific promoter effect. Result ? about the same
What does measuring flourescence get them ? protein abundance. Does this get mRNA levels ? No.
Test multiple ways of measuring flourescense -> all same. Show figure S4.
Also test western blot ? separate proteins on gel and use antibodies to identify
Can measure "free energy" of molecule. Basically how much more stuff could it do. If it's 0, that means it's pretty stable and can't do anything more. Show figure 2. RNA folding.
CAI nor freq. optimal codons correlate w/ fluorescence. (Why use multiple measures ? )
Rare codons, rare pairs, also no correlation. Nor did CAI of first amino acids. Figure 2A.
Conclusion: nada
Ask if position of codon makes a difference. For basic codon usage model, should it ? Figure S6
Suggests mRNA structure.
Measure free energy. And ? total was not correlated but folding of first 1/3 was. Fig. 2B and 2C.
Sliding windo analysis showed first part explained most of variation under both promoters.
Conclusion: tightly folded obstructs initiation of translation. Where else do we see hairpin loops ? Transcription termination and initiation in tryp operon.
If blocking initiation, why didn't Shine-Delgarno sequence show up ? (what is S-D ? region just upstream of translation start site for ribosome binding) : their sequences all had same S-D!
mRNA levels as measure by Northern blot, but variaiton 3X less. Why ? codons affect transcription less than translation. But still correlated w/ flourescence (more mRNA -> more protein) and affected by mRNA folding. What's interpretation ? mRNA folding affects ribosome binding. less ribosome = less translation, also = more exposure to digestion, so mRNA lasts less time so observe fewer transcripts.
Prediction: adding weak 5' mRNA folding sequence should increase expression even if CAI not changed. Fig. 3
CAI = faster growth! mRNA folding noT! Why ? Slower translation uses up ribosomes, so overall cell process has to wait longer.
Conclusions: So instead of high CAI -> high expression (because fast); high expression -> high CAI (because you don't want to use up all the ribosomes)