Question on transcription start site and promoter cloning - (Mar/14/2008 )
I'd like to ask whether the Transcription start site (+1) is at the first nucleotide of the 5'UTR?
I have cloned the promoter of a gene spanning at position -1900 to -1 (relative to +1 according to my above interpretation) into the pGL3-basic luciferase reporter vector. I have co-transfected this reporter vector together with a TF expression vector(with GFP) or an empty expression vector in a ratio of 5:1 into HeLa cells. However, the Relative activity activity of cells w/ or w/o TF remains around 0.6-0.8 if the RLA of the empty reporter vector =1.
I m wondering whether the promoter I cloned do not have transcriptional activity at all, will it likely be the case?
Plx give me some opinion.
Thx in advance.
2. several ways of looking at it. (a) no transcriptional activity, tough luck ( wrong cloning direction, check strategy © presence of a strong repressor in this fragment, do Transfac analysis, clone several truncated fragments (d) promoter may be active only in the genomic context, get stable cell line (e) this promoter does not work in HeLa cells, change cell line (f) there was a technical mix-up, check/throw away everything and repeat (g) some of the above or some more..
Some additional points to be considered:
- How about just cloning the core promoter for example a 200-300 bp piece because the upstream sequence may contain inhibitory elements or regions.
- The promoter sequence may not just be the sequence upstream the TSS, but also including sequence after the TSS. So your insert should include for exmaple 50-100 bp sequence after the TSS. I previously clones a promoter containing over 100 bp sequence of the first exon and obtained very high promoter activity.
- Make sure there is no alternative promoter usage, alternative exons further upstream the putative TSS. The best way of checking that is to use the UCSC or Emsembl genomoe broswers
The activity of most basal promoters is improved by including some of the sequence downstream of +1. Most basal promoters include this downstream region. It also could be that your promoter has low expression levels to begin with. One thing not suggested by others that i would do is research the promoter. Look at what other papers have done, particularly the paper characterising the promoter for the first time. Then you can see exactly what sequence is required for good expression or if expression levels are low to begin with.