Promoter_Characterization

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Prokaryotic Promoters
M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
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Prokaryotic Genes

• Recall - prokaryotes have a single circular
  chromosome
• Also, no cell nucleus, and no introns
• Therefore, prokaryotic gene structure is quite
  simple

Translational start site (AUG)
Translational stop site
Promoter region
Open Reading Frame
Transcriptional start site
Operator sequence
Transcriptional stop site
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Prokaryotic Operons
Operon structure
Downstream
Upstream
Promoter
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.
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Bacterial Gene Signals
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Gene 2
Gene 1
Bacterial genomes have simple gene structure

• - 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.

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Promoters

• 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.

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Prokaryotic promoter
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Typical E. coli Promoters
TATAAT
Pribnow Box
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Consensus sequences of E. coli Promoters
T80A95T45A60A50T96

• the sequence at the promoter can regulate
  efficiency of initiation
• different sigma factors may associate with RNA
  polymerase, which target specific promoters

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Methods for characterization of promoters

1. DNAse protection method
2. DMS protection method
3. Foot-printing method

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1. DNase Protection method

• The region of DNA in contact with RNA polymerase
  can be isolated
• Allow the piece of DNA containing the promoter to
  interact with RNA polymerase
• Treat with DNase I
• Dissociate the enzyme and isolate the DNA
• Determine the size by gel electrophoresis
• Determine the sequence by standard method

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_ _ _ _ _ _
_ _ _ _ _ _
DNase I

Mono and dinucleotides
DNA molecule with promoter
RNAP DNA complex
RNAP
Dissociate DNA from enzyme
Sequence the Promoter DNA
Promoter region
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2. DMS Protection method

• 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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DNA molecule with promoter
RNAP
DMS
_ _ _ _ _ _
_ _ _ _ _ _











Mono and dinucleotides
RNAP DNA complex
Methylated purines
Dissociate DNA from enzyme
Sequence the Promoter DNA
Promoter region
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3. Foot-printing method

• Take a DNA fragment with known Restriction sites
• Dephosphorylation Alkaline phosphatase
• End labelling 5 is to be labelled with
• gamma-32P- ATP using T4 polynucleotide
  kinase
• Remove a small fragment by RE digestion
• Allow the labelled DNA to interact with RNA
  polymerase
• - One sample is to be maintained without
  RNAP
• treatment
• Using DNA endonuclease briefly digest the DNA
  sample treated with RNAP Nicking occurs
  randomly at all places except those protected by
  RNAP
• Analyze both the samples (with and without RNAP
  interaction) following agarose gel electrophoresis

A method to detect where a protein binds to DNA
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(No Transcript)
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(No Transcript)
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Foot-printing
One end labelled DNA
RNAP
Used extensively for mapping contact points
between promoter sequences and RNA polymerase
and/or regulatory proteins
No RNAP
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Interpretation

• 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
  nx different sizes of DNA fragments will be
  represented
• The positions of the missing bands are the
  positions of the n bands on DNA

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THANK YOU