This post is not made by me, it is made (collected) by my friend and I want to share it with you, thanks :)
DNA sequences in the vicinity of the structural portion of a gene that are required for gene expression.
As we have mentioned several genes seem to be transcribed coordinately. For example, the different members of a storage protein or photosynthetic protein family are expressed at the same time in development. These genes have sequence modules in common that control the coordinate regulation. These modules are called response elements. These elements are a class of cis-acting elements.
Features of Response Elements
- contain short consensus sequences
- modules are related but not identical
- not fixed in location but usually within 200 bp upstream of the transcription start site
- a single element is usually sufficient to confer a regulatory response
- can be located in a promoter or an enhancer
- assumed that a specific protein binds to the element and the presence of that protein is developmentally regulated
Trans-acting factors - factors, usually considered to be proteins, that bind to the cis-acting sequences to control gene expression
We have not discussed trans-acting factors yet. What are some of the properties of different trans-acting factors:
- subunits of RNA polymerase
- bind to RNA Polymerase to stabilize the initiation complex
- bind to all promoters at specific sequences but not to RNA Polymerase (TFIID factor which binds to the TATA box)
- bind to a few promoters and are required for transcription initiation; these are positive regulators of gene expression
Those factors which bind to consensus module sequences can bind to any promoter that contains the sequence. The binding of multiple factors, for example, multiple trans-acting factors each with one of the four properties mentioned above, may be essential for transcription initiation. Enhancers, which normally have a consensus 72 bp repeat sequence, have sites for multiple trans- acting factors to bind. Thus genes with enhancers may require several complexes to be constructed for gene expression to be initiated.
Functions Of Transcription Factors
- recognize target sequences in DNA
- interact with other transcription factors
These trans-acting factors can control gene expression in several ways:
- factor may be expressed in a specific tissue manner (spatial regulation)
- factor may be expressed in at specific time in development (temporal regulation)
- factor may require modification (phosphorylation)
- factor may be activated by ligand binding
- factor may be sequestered until an appropriate environmental signal allows it to interact with the nuclear DNA
Each transcription factor has a characteristic motif. These motifs are short structures which comprise only a small portion of the protein. These motifs are used to define a specific class of trans-acting factors. The following are specific examples of trans-acting factors, each with a defined and specific motif.
- steroid receptors
- zinc finger proteins - binds at a specific site
- leucine zipper proteins - leucine appears every seven amino acids in a region of the protein
We now need to see how experiments are performed which determine that a trans-acting factor binds to a promoter or enhancer. The principle technique is called gel retardation assay. The principle underlining this procedure is that a DNA molecule exhibits a slower mobility when it is bound by a protein than when it is naked. These experiments are performed on a polyacrylamide gel under low ionic conditions.
Steps of gel retardation assay
- Identify a promoter region of interest.
- Make a crude nuclear protein extract from the developmental stage that the gene of interest is
- being expressed.
- Mix the promoter fragment of interest with the crude extract.
- Run the following samples:
- a. free promoter fragment
- b. promoter fragment + crude extract mixture
- c. crude extract + competitor + promoter fragment
Copyright © 1998. Phillip McClean