Question

Transcriptional initiation at defined sequences in DNA is required to ensure that the 5' end of the RNA transcript is correct. Describe the two types of experimental approaches that have been used to identify regulatory DNA sequences located near gene start sites.

Answer 1

Regulatory DNA sequences near gene start sites can be identified using the following approaches:

1. Mutating the regulatory sequences and then checking the binding of the RNA polymerase or other transcription factors using invitro technique EMSA and in-vivo technique chromatin immunoprecipitation. Using these techniques one can find out whether mutating or removing the regulatory DNA sequence abrogates the binding of necessary transcription factors for initiation of transcription.

2. Some part of the DNA sequence near the start site can be fused to a reporter gene. Many constructs can be made for this fusion by mutating several residues of the regulatory DNA and then reporter assay can be performed for all the constructs to find out or identify the regulatory sequence. Reporter genes such as GFP, YFP, or mCHERRY can be used or otherwise yeast one hybrid beta-galactosidase assay can be utilized for this.