promoter characterization
DESCRIPTION
BIOCHEMISTRY - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/1.jpg)
M.Prasad NaiduMSc Medical Biochemistry,
Ph.D,.
![Page 2: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/2.jpg)
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
![Page 3: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/3.jpg)
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.
![Page 4: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/4.jpg)
- 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
![Page 5: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/5.jpg)
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.
![Page 6: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/6.jpg)
Prokaryotic promoter
![Page 7: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/7.jpg)
TATAAT
Pribnow Box
![Page 8: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/8.jpg)
‘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
![Page 9: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/9.jpg)
1. DNAse protection method2. DMS protection method3. Foot-printing method
![Page 10: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/10.jpg)
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
![Page 11: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/11.jpg)
_ _ __ _ _
_ _ __ _ _
+
DNA moleculewith promoter
RNAP RNAP +DNA complex
Mono and dinucleotides
DNase I
Dissociate DNAfrom enzyme
Promoterregion
Sequence thePromoter DNA
![Page 12: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/12.jpg)
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
![Page 13: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/13.jpg)
_ _ __ _ _
_ _ __ _ _
+
RNAP +DNA complex
Mono and dinucleotides
DMS
Dissociate DNAfrom enzyme
Promoterregion
Sequence thePromoter DNA
DNA moleculewith promoter
RNAP
** *
**
***
* *
Methylatedpurines
![Page 14: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/14.jpg)
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
![Page 15: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/15.jpg)
![Page 16: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/16.jpg)
![Page 17: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/17.jpg)
Foot-printingOne end labelled DNA
Used extensively for mapping contact points between promoter sequences and RNA polymerase and/or regulatory proteins
RNAP
No RNAP
![Page 18: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/18.jpg)
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
![Page 19: PROMOTER CHARACTERIZATION](https://reader033.vdocument.in/reader033/viewer/2022050908/56812a30550346895d8d2d93/html5/thumbnails/19.jpg)