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