I am trying to characterise the promoter of a particular gene. Ncbi already gives the Sequence of the 5'utr, only part of it I am guessing. ensemble, ucsc and epd gives the probable tss. This gene has multiple variants having different tss possibly and thus controlled by different promoters. I want to experimentally characterise one of the variants' promoters if not all. How much sense does it make to do 5' race, primer extension and nuclear run off assays??? I am aware that in the end I would have to create promoter constructs and check their firing and determine regions absolutely required for transcription. But how to determine which promoter controls which variant?? What is the tss of that variant??
Do you get two transcripts from the gene? In this case, you have to eliminate both possible peudogene products unprocessed and other enhancer sequences found in the upstream and downstream regions of the gene. Also need to eliminate enhancer sequence in the middle of the gene having alternative transcription products. Or you found alternative TSS in the partial promoter sequence. In both case, make sure first that there is no alternative splicng of the particular gene.
5'-RACE worked well after several rounds of hiccups in our case of cloning 2kb promoter. NRO assay make sense, too. Just before start, you should have evidences for that different gene promoters exist or just presence of an intragenic ehanacer sequence in the middle of the gene in which you would get two variant transcripts.These elements are operating, in particular, in developmental gene expressions.
Good luck!
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