How much does it matter to ligate coding sequences with upstream regions (including promoters, etc) without scar?
I'm always very cautious about that, trying not be bring restriction sites between the coding sequence and the promoter region (from -1 site to the upstream sequence) when ligating them. Does it matter so much?
I know that the RBS in bacteria are very important and conserved. Many experiments does not seem to care about it and use restriction assembly, why?
These days I've been working with a yeast (eukaryotic). I haven't checked much, but also know some reports about the importance of the -3 site on efficient expression. Still, most people use restriction assembly. I'm in doubt that using that, the translation efficiency will be affected. The scanning model of eukaryotic translation doesn't explain why translation initiation is affected by adjacent regions of AUG.
The AUG should be in a strong consensus sequence for translation initiation, otherwise you will have a high rate of read-through.
You can read a bit about it here: http://en.wikipedia.org/wiki/Kozak_consensus_sequence
Very helpful. I'm much clearer about it now.
Sorry about the ambiguity of the meaning of +1, but it seems that both of these two designation happens depending on the context.
I would believe that the context sequence of AUG does play a role.
Article Jackson RJ, Hellen CU, Pestova TV.. The mechanism of eukaryo...
More is to be revealed by structural biologists about initiation codon recognition, but now from the occurrence of consensus sequence and results of mutational experiments, I assume the sequence context is important. In fact, the promoter region (just upstream of the ATG site) displayed high similarity with the S.cerevisiae consensus sequence near the ATG site, though the promoter is from another yeast.
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