PROMOTER CHARACTERIZATION
-
Upload
mprasadnaidu -
Category
Documents
-
view
40 -
download
3
description
Transcript of PROMOTER CHARACTERIZATION
M.Prasad NaiduMSc Medical Biochemistry,
Ph.D,.
Recall - prokaryotes have a single circular chromosome
Also, no cell nucleus, and no introns Therefore, prokaryotic gene structure is quite
simple
Transcriptionalstart site
Promoterregion
Operatorsequence
Open Reading Frame
Transcriptionalstop site
Translationalstart site (AUG)
Translationalstop site
Downstream
Operon structure
Promoter
Upstream
Gene 1 Gene 2 Gene 3
In prokaryotes, sometimes genes that are part of the same operational pathway are grouped together under a single promoter. They then produce a pre-mRNA.
- Promoter
-35 sequence (T82T84G78A65C54A45) 15-20 bp
-10 sequence (T80A95T45A60A50T96) 5-9 bp (Pribnow Box)
-Start of transcription : +1 initiation start: Purine90
-Translation binding site (Shine-Dalgarno) 10 bp upstream of AUG (AGGAGG)
- One or more Open Reading Frame
•Start-codon: ATG (unless sequence is partial)
•stop codon for gene 1 ..
Separated by intercistronic sequences.
Gene 1 Gene 2Bacterial genomes have simple gene structure
+1
Promoter sequences facilitate the binding of the RNA polymerase to the DNA to be transcribed.
Promoters of different genes have distinct sequences, although most have characteristic short sequences of 6 to 10 bases at a position between 10 to 30 nucleotides upstream -10 sequence: Hexamer: TATAAT – Pribnow Box
(Pribnow, 1975) and -35 sequence, an hexamer : TTGACA in prokaryotes.
Prokaryotic promoter
TATAAT
Pribnow Box
‘Consensus’ sequences of E. coli Promoters
mRNA start pointPribnow box
• the sequence at the promoter can regulate efficiency of initiation
• different sigma factors may associate with RNA polymerase, which target specific promoters
-35 region
T80A95T45A60A50T96
1. DNAse protection method2. DMS protection method3. Foot-printing method
The region of DNA in contact with RNA polymerase can be isolated
1. Allow the piece of DNA containing the promoter to interact with RNA polymerase
2. Treat with DNase I3. Dissociate the enzyme and isolate the DNA4. Determine the size by gel electrophoresis5. Determine the sequence by standard
method
_ _ __ _ _
_ _ __ _ _
+
DNA moleculewith promoter
RNAP RNAP +DNA complex
Mono and dinucleotides
DNase I
Dissociate DNAfrom enzyme
Promoterregion
Sequence thePromoter DNA
Specific points of contact within the contact region can be identified
Dimethyl sulphate methylates N3 of A or N7 of G, but not C or T
Glycosidic bond of methylated As or Gs is unstable and can be broken by heating at neutral pH leaving deoxy ribose from the chain – DNA degradation results
Region of the DNA bound by RNA polymerase will not be methylated – it will be intact
Dissociate the enzyme and isolate the DNA fragment that corresponds to the promoter
_ _ __ _ _
_ _ __ _ _
+
RNAP +DNA complex
Mono and dinucleotides
DMS
Dissociate DNAfrom enzyme
Promoterregion
Sequence thePromoter DNA
DNA moleculewith promoter
RNAP
** *
**
***
* *
Methylatedpurines
3. Foot-printing method
1. Take a DNA fragment with known Restriction sites2. Dephosphorylation – Alkaline phosphatase3. End labelling – 5’ is to be labelled with gamma-32P- ATP using T4 polynucleotide kinase4. Remove a small fragment by RE digestion5. Allow the labelled DNA to interact with RNA polymerase - One sample is to be maintained without RNAP treatment6. Using DNA endonuclease briefly digest the DNA sample
treated with RNAP – Nicking occurs randomly at all places except those protected by RNAP
7. Analyze both the samples (with and without RNAP interaction) following agarose gel electrophoresis
A method to detect where a protein binds to DNA
Foot-printingOne end labelled DNA
Used extensively for mapping contact points between promoter sequences and RNA polymerase and/or regulatory proteins
RNAP
No RNAP
If the DNA contains ‘n’ bp and RNAP is not added, ‘n’ sizes of DNA fragments will be present
However, if RNAP binds to ‘x’ bp and thereby prevents access of the DNA to the nuclease, only ‘n–x’ different sizes of DNA fragments will be represented
The positions of the missing bands are the positions of the ‘n’ bands on DNA