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

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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

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+

RNAP +DNA complex

Mono and dinucleotides

DMS

Dissociate DNAfrom enzyme

Promoterregion

Sequence thePromoter DNA

DNA moleculewith promoter

RNAP

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**

***

* *

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