[Overexpression] Without upstream transcription terminator - (May/09/2017 )

Hi all,

I have been told that during the cloning of gene for protein overexpression, there should be a terminator placed upstream of a promoter and gene, if there is another gene (e.g. resistant marker) upstream to the target gene. Otherwise, the expression of the target gene will be affected.

However, is that real?

Yes, it is real. You also need the terminator to ensure that the protein is correct when expressed - otherwise transcription just continues and you will end up with a large nonsensical protein expressed from the bases 3' of the intended sequence.

Thank you very much pulchella,   

However, how could the upstream promoter affect the downstream promoter if a terminator is not located in between? What is the mechanism of this?

bob1 on Tue May 9 13:54:24 2017 said:

Yes, it is real. You also need the terminator to ensure that the protein is correct when expressed - otherwise transcription just continues and you will end up with a large nonsensical protein expressed from the bases 3' of the intended sequence.

It's called transcriptional run-through - basically the transcriptional machinery will keep transcribing the bases beyond where you want (or should) have them terminate, meaning that you get nonsensical expression of the bases between the upstream and downstream genes. Without an internal ribosome entry site (IRES) this will prevent separate transcription of the downstream gene.

bob1 on Tue May 16 18:04:12 2017 said:

It's called transcriptional run-through - basically the transcriptional machinery will keep transcribing the bases beyond where you want (or should) have them terminate, meaning that you get nonsensical expression of the bases between the upstream and downstream genes. Without an internal ribosome entry site (IRES) this will prevent separate transcription of the downstream gene.

Thank you very much Bob, it is very much helpful