INDM 3007

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INDM 3007

• Gene expression and regulation
• Dr. Patrick Caffrey
• Dr. Wim Meijer

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Flow of information
DNA double stranded helix of desoxyribonucleotide
s
RNA Single stranded ribonucleotide chain
Protein Amino acid chain
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DNA is a double helix of anti-parallel DNA
strands. Each strand contains four nucleotides
dA,dC,dT,dG
DNA is complementary both strands contain
information
Only one strand is transcribed into RNA
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Remember that DNA/RNA has a direction 5--gt 3
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5
3
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Transcription from DNA to RNA
Catalysed by RNA polymerase a multimeric enzyme
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Structure of RNA polymerase holoenzyme at 28Å
resolution
This is what it really looks like.
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Tasks performed by RNA polymerase

• Binds to specific sites of DNA promoter regions
• Unwinds the promoter
• Copies DNA into RNA
• Catalyses the formation of phospho diesterbonds
• Stops transcribing at a specific site terminator
• RNA is released from DNA
• RNA polymerase releases from DNA

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RNA polymerase exists in two forms
Holo enzyme Sigma, 2 alpha, beta beta
subunits Core enzyme 2 alpha, beta, beta
subunits
Core enzyme contains enzymatic activity it makes
RNA but does not bind to a specific nucleotide
sequence
Strong aspecific DNA binding is essential to
transcribe long stretches of DNA RNA polymerase
is processive
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The sigma factor confers promoter specificity to
RNA polymerase
Holo RNA polymerase binds very strong to the
promoter, holoenzyme transcribes 2-9 bp, then
releases RNA and begins again abortive
transcription.
RNA polymerase is unable to clear the promoter
Sigma factor is therefore released after
initiation of transcription
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Only one strand of DNA, the sense strand, is
transcribed into RNA.
DNA is partially unwound by RNA polymerase to
gain access to the Sense strand
Formation of the transcription bubble
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Transcription termination
Two forms of termination Rho independent of
intrinsic termination Rho dependent termination

• RNA polymerase pauses stops adding
  ribonucleotides to RNA chain
• Releases RNA from DNA
• Releases from DNA
• Information for termination is contained in RNA
  not DNA

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Rho independent termination
Due a GC rich inverted repeat, a stable hairpin
structure forms in RNA RNA polymerase pauses at
hairpin structure RNA-DNA pairing, especially at
UA basepairs is unstable RNA and RNA polymerase
release
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Rho dependent termination
Rho factor is a protein containing helicase
activity unwinding nucleotide helices, in this
case RNA-DNA hybrids
Rho binds recognition site Follows RNA
polymerase, and catches up when it pauses at
hairpin Helicase activity unwinds RNA-DNA
hybrid RNA pol releases
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An implication of the hot-pursuit model
translating ribosomes may block rho from reaching
RNA polymerase. Result termination does not
occur
Stalled RNA polymerase
Rho
Ribosome