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Alternative start codon in plasmid


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#1 tapi

tapi

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Posted 28 October 2013 - 07:24 AM

Hi all,

 

just a question of concept:

 

We made a mistake in the designing of a construct but we realized once the experiment was finished.

 

We wanted to overexpress a gene. We cloned the desired gene substituting a LacZ gene but we accidentally left a start codon (with kozak sequence) upstream of the ATG of our gene (which also have its own Kozac sequence). The ATG left by mistake was from a hp68/70 promoter-LacZgene

 

This not desired ATG codon was in frame with the other and there was a stop codon between both of them.

 

Surprisingly everything worked perfectly and the protein seems to give the effect desired.

 

I was wondering what has happened here. Possible explanations could be:

 

1- Translation started in alternative ATG and ended and later started again with the correct ATG?: I though that the mRNA deattach from ribosome after stop sequence is found

 

2- Translation stated in the alternative ATG but continued because the stop codon was not enough to stop translation, resulting in a protein with some more aminoacids at the beginning but perfectly functional?: Maybe there is any other more sequence required to stop translation apart from the stop codon?

 

I am extremely puzzled by this and I would really appreciate some comments

 

Many thanks!!!






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