Physiology 3140A Lecture Notes - Lecture 3: Dna Binding Site, Dna Construct, Dna-Binding Domain
22 May 2018
School
Department
Course
Professor
Physiology 3140
Dr. Pin
Transcription II
Why would you want to know the promoter and enhancer regions? Why would you want to know what
regulates the gene?
A: the underlying cause of every disease is a dysregulation either in the function or expression of a gene.
So if you know what regulates the gene, you know how to counteract that negative impact. If you know
the sequences that regulate the gene and do a sequence analysis of individuals with that disease, you can
find out where that mutation is and if its in a region that is involved in its expression, that can help as
well. Also, if you are trying to develop a therapy. If you want for ex: EGF (growth factor involved in many
processes) expressed and you want it to fix the cell. So how can you restrict the expression of this (bc if
you allow the protein to be expressed everything, it will affect every cell type). So if you want something
very specific, you want to be able to drive that expression only in the tissue of interest.
PROMOTER ANALYSIS
Why is it important?
- May determine the factors that promote expression
- May aid in identify genetic mutations that have a distinctive phenotype but are not within a gene
- Can be used as a tool to drive the expression of other genes in a cell, tissue and time specific
fashion
- Knowing these promoters is a good research and therapeutic tool
- E.g. Viral introduction for gene therapy
Questions from last years exam:
- This is 4 different regions of a promoter, with an enhancer and repressor elements that are
driving the expression of this reporter gene, luciferase
- What’s DNA construct would give the highest expression?
o B – lacks a repressor domain that would reduce expression
- What DNA construct would give the lowest expression?
o D – lacks a tissue specific transcription factor binding site within the promoter so
enhancer or repressor will not function that well
- THIS IS GOING TO BE AN EXAM QUESTION!!!!
TRANSCRIPTION FACTORS
How would you define a transcription factor?
- Over 2,000 transcription factors identified to date
- protein that controls the rate of transcription of genetic information from DNA to mRNA, by
binding to a specific DNA sequence
- molecule that controls the activity of a gene by determining whether the gene’s DNA is
transcribed into RNA
find more resources at oneclass.com
find more resources at oneclass.com
- protein involved in the process of converting, or transcribing, DNA into RNA
- so transcription factors DON’T (AVE to have binding of DNA
o 90% of the time it will, but it doesn’t have to
General Transcription Factors
- initiates gene expression
- TFII proteins
- Bind all genes
- RNA polymerase II is NOT considered a transcription factor
Specific Transcription Factors
- increases transcription rate and efficiency for a specific gene
- only binds to a specific gene but is required for complete expression of a gene
- show both time specificity and tissue specificity
What would the perfect transcription look like?
- DNA binding domain
o Has basic residues (the +ive charge is going to allow it to be able to interact)
- Transcriptional activation domain/repressor domain (depending on whether its going to turn
on or off transcription)
- Nuclear localization signal (NLS) – brings the protein into the nucleus
- Ligand binding domain
- Domains for binding co-activators or co-repressors
- Binding domain for other TFs
- Not all transcription happening has all of these
E2F Transcription Factor 1
- Expressed in almost every cell
- Involved in cell cycle regulation
- There is all kinds of binding domains in here
- NLS (within the cyclin A binding domain)
- DNA binding domain (basic residues that interact with the DNA)
- A domain that will activate transcription
o This is the region of the protein that brings in and interacts with the polymerases,
general transcription factors
- Binding sites for other TFs
o Transcription factors usually work as multimers (not usually a single protein)
- Ability to interact with other co factors (interaction with co-activator)
o In the CBP/p/CAF is a protein that will interact with E2F1 and that protein will modify
the chromatin (its an active genetic regulator)
o This region really allows it to open up the DNA
- There are phosphorylation sites that regulate its expression and its ability to be stable
(sometimes phosphorylation causes degradation, other times it makes it inactive/active)
find more resources at oneclass.com
find more resources at oneclass.com
Groups of Transcription factors are often classified based on their protein binding motifs
- These regions that are used to separate the classes…
o Function very similarly
o They will bind to common DNA regulatory elements
o )t’s the other parts outside of those domains that indicate specificity
Super Classes:
1. Basic
o Proteins that bind DNA through this basic region
o Either have a…
▪ Leucine zipper (ZIP)
▪ Helix-loop-helix (HLH)
▪ HLH-ZIP (have both)
o The rest of the region is involved in the dimerization of the proteins – so these TFs work
as dimers
o HLH proteins bind the consensus DNA sequence CANNTG (palindromic)
▪ Most DNA consensus sequences, if they are binding dimers are palindromic
o This sequence happens every 250 bp within the genome –
o SO these proteins require a consensus sequence outside of this basic helix loop helix
region – there is going to also be something else there
o Bc if its going to bind every 250bp, how does this dictate specificity?
o This consensus sequence is the middle of it, and there is also going to be other parts of
the protein that is going to be cell-specific
o These 2 proteins (dimer) sit on the DNA in a way that there is one on either side – this
can happen bc the consensus sequence is palindromic
o Some of these work as homodimers (2 proteins are the same) but most of the time they
are heterodimers
▪ There is 2 classes of these heterodimers
• Class A protein: almost expressed everywhere
• Class B protein: dictate the specific expression in the tissue
2. Zinc-coordinating DNA binding domains
o Zinc fingers or zinc clusters
o They come as pairs
o Require Zn for their ability to work (they have a binding region that can bind the Zn
then bind the DNA sequences)
o Provide specificity for DNA sequences
o They have a helical turn and a particular looping structure, which is the consensus
sequence
o Its consensus sequence: F/Y-X-C-X-X-F/Y-X-X-X-X-F/Y-X-X-X-X-X-X-X-X-H-X-X-X-H-
▪ So in this consensus sequence, its not that important what the X’s are but its
more important the distance bw the F/Y and C’s
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
A: the underlying cause of every disease is a dysregulation either in the function or expression of a gene. So if you know what regulates the gene, you know how to counteract that negative impact. Also, if you are trying to develop a therapy. If you want for ex: egf (growth factor involved in many processes) expressed and you want it to fix the cell. So how can you restrict the expression of this (bc if you allow the protein to be expressed everything, it will affect every cell type). So if you want something very specific, you want to be able to drive that expression only in the tissue of interest. May determine the factors that promote expression. May aid in identify genetic mutations that have a distinctive phenotype but are not within a gene. Can be used as a tool to drive the expression of other genes in a cell, tissue and time specific fashion.
