DNA Regulatory Sequence Analysis / DNA Binding Protein Analysis

Gene regulation is a tightly controlled and regulated process. Gene regulation is complex process and involves various control checkpoints. Promoters, enhancers, DNA binding proteins plays major role in gene regulation.

•         Transcription of gene is controlled / regulated by Promoter and enhancers.

•         The activity of enhancers and promoters are controlled by Transcription factors (the DNA binding Proteins).

•         The DNA binding proteins plays a major role in the gene regulation, which can be identified by the following methods.

Methods for Identifying DNA binding Proteins / Gene Regulation

Following are some of the methods used for identification and analysis of gene regulation

•         DNA Foot printing – Identifies site at which the protein binds.

•         Gel Shift Assay – Identifies DNA-Protein Complex.

•         CAT Assay – Measures Transcriptional Activity.

DNA Foot Printing

In DNA Foot Printing method,

The 5’ end of cloned DNA fragment having the promoter or enhancer to be studied is radiolabeled. 

The labeled DNA fragments are then divided into 2 sets. The first is set is incubated with nuclear extract having DNA binding proteins and the second set is used as control without incubating. 

After the incubation, both sets are treated with nucleases. Nucleases cleave the DNA into smaller fragments. 

Nucleases cannot cleave the regions where the protein is bound. The nuclease treated products are run on gel and autoradiographed.

By comparing the migration patterns of control Sites at which the DNA binding proteins interacts can be identified. The X-ray film gives the DNA foot print.

  

Gel Shift Assay

In Gel Shift Assay method,

DNA migration under electric field is used to identify the DNA binding proteins interaction. The migration of DNA-Protein complex is much lesser than the DNA alone. The radiolabeled DNA fragments are incubated with nuclear extract having DNA binding proteins, after incubation DNA fragments are run on gel along with the control (Not Incubated).

 By looking at the autoradiographic X-ray film DNA-Protein complex can be identified.

CAT Assay

In CAT Assay method,

Reporter gene is cloned with the promoter to be studied. The promoter-reporter construct is introduced into the cell for analyzing the transcriptional level. Active Promoter will initiate transcription of the reporter gene and the level of product formation can be analyzed.

Mostly used reporter genes:

•         CAT gene – Chloramphenicol Aceyl Transferase.

•         Luciferase gene.

•         Expression levels of these genes are used to analyze the promoter.

References:

Lehniger, Biochemistry

J.D Watson, Recombinant DNA

Kuby, Immunology