Hi Riddhi, PlantCare software will only tell you which putative cis-elements you have present in your promoter, not the actual TFs. To identify the TFs that bind to your sequence you will have to perform techniques such as Yeast One hybrid. I believe that will be the best approach to identify TFs that bind your promoter region.

Hi Riddhi, PlantCare software will only tell you which putative cis-elements you have present in your promoter, not the actual TFs. To identify the TFs that bind to your sequence you will have to perform techniques such as Yeast One hybrid. I believe that will be the best approach to identify TFs that bind your promoter region.

Thank you Tiago for your help.

Hi Riddhi,

As was suggested by Tiago, you could use yeast one-hybrid. This is the low-budget method of choice. However, theer are some problems when working with a 'full promoter' sequence:

1) like many heterologous systems, you might get a lot of false positives or candidates that work only because of yeast transcription factors (TFs)  that assist the plant hybrid-TFs in actvation. Hence, good candidates in yeast might not function in plants, because a co-factor is missing (at that point in time or due to tissue specific expression)

2) Usually sequences longer than ~300 do NOT work properly in yeast one-hybrid. Either there is no activation or very little = of background/control

3) Most researchers will first narrow a promoter to regions of interest that are not longer than 20 - 50 bp, which have been tested for a certain function in plants, e.g. in transiently transfected protoplasts or in promoter::reporter (GUS) assays. These smaller fragments are multimerized (usually 4x) for yeast one-hybrid so that a putative binding site occours more often in the reporter vector, which gives higher activation in yeast and, hence, a better growth.

4) Yeast one-hybrid - like any other experiment - depends on high quality controls. You need to start with the generation of native and mutated-binding site reporter strains. And you should have both as non-transformed control strains. Your screen is over, as soon as you find yeast growing on your negative control plates - which happens after 2-4 weeks incubation.

You might like to read further (Wanke & Harter, 2009: Analysis of plant regulatory DNA sequences by the yeast-one-hybrid assay. Methods Mol Biol.)

As you are working at a chemical institute: The high budget method is:

Use your promoter as pray and incubate with cell extracts. TFs will bind and you could analyse them via LC-MS/MS.

DNA-centric Yeast-1-hybrid is the best way to discover TFs or othe proteins that bind to promoter

Thank you so much