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Dr. Parvin Pasalar
Tehran University of Medical Sciences
دانشگاه علوم پزشكي وخدمات بهداشتي درماني تهران
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Objectives: To know and explain: What are needed for transcriptionWhat are needed for transcription Template Building blocksBuilding blocks EnzymesEnzymes EnergyEnergy Protein Factors Protein Factors Different Stages of Different Stages of transcriptiontranscription Initiation (promoter recognition) Elongation Termination Differences between transcriptiontranscription in eu and
prokaryotes Toxins and antibiotics that inhibit
transcription
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DNA
mRNA
Transcription
Prokaryotic TranscriptionProkaryotic Transcription
Cell
Polypeptide(protein)
TranslationRibosome
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What are needed for What are needed for transcriptiontranscription
PermissionPermission Template (DNA, Gene)Template (DNA, Gene) Building blocks (4 types of Building blocks (4 types of
NTPs)NTPs) EnzymesEnzymes Energy (ATP and the other Energy (ATP and the other
NTPs)NTPs) Protein factors (Protein factors (σσ & & ρρ))
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5’ 3’
RNA Transcript
A “Simple” Prokaryotic A “Simple” Prokaryotic GeneGene
Protein Coding Region
Terminator Sequence
Promoter/Control Region
Transcription Start Site
5’ 3’AUG UAA
3’ 5’
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Promoter & itsPromoter & its consensus sequence consensus sequence determinationdetermination
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Holoenzyme: 2 ’ for initiation Core enzyme: 2 ’ for elongation
E. coli RNA polymerase
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Different stages of Different stages of TranscriptionTranscription
Recognition & InitiationRecognition & Initiation
ElongationElongation
TerminationTermination
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Initiation: close and open Initiation: close and open complexcomplex
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The elongation stageThe elongation stage
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RNA chain terminationRNA chain termination Termination occurs at Termination occurs at
terminator DNA terminator DNA sequences. sequences.
1. Rho-independent: the 1. Rho-independent: the most common stop most common stop signal by RNA hairpin signal by RNA hairpin formation(self-formation(self-complement structure)complement structure)
commonly GC-rich to commonly GC-rich to favor the structure favor the structure stabilitystability
2. Rho-dependent 2. Rho-dependent Termination: RNA Termination: RNA wraps around Rho wraps around Rho hexamer and stops hexamer and stops transcription. transcription.
Inverted repeats
ρ- independent
ρ- dependent
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RNAPol.
5’RNA
RNA Pol.
5’RNA
TerminationTerminationRho IndependentRho Independent
Terminator
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RNAPol.
5’RNA RNA
Pol.
5’RNA
TerminationTerminationRho IndependentRho Independent
Terminator
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RNAPol.
5’RNA
TerminationTerminationRho DependentRho Dependent
Terminator
RNA Pol.
5’RNA The terminator
sequence slows RNA polymerase
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RNAPol.
5’RNA
TerminationTerminationRho DependentRho Dependent
Terminator
Help, rhohit me!
RNA Pol.
5’RNA
Rho catches up with RNA polymerase
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RNAPol.
5’RNA
TerminationTerminationRho DependentRho Dependent
Terminator
RNA Pol.
5’RNA
The elongation complex disintegrates
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A “Simple” Eukaryotic A “Simple” Eukaryotic GeneGene
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DNA
Cytoplasm
Nucleus
Eukaryotic geneEukaryotic gene
ExportG AAAAAA
RNA
Transcription
Nuclear pores
G AAAAAA
RNAProcessing
mRNA
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Different Eukaryotic RNA polymerases
Basis of classification:1- Elution at different salt concentrations during ion-exchange chromatography2- Different sensitivity to α amanitin
Pol I: very insensitive to α amanitin, transcribes the gene for pre-rRNA (28S, 5.8S, 18S)
Pol II: very sensitive to α amanitin, transcribes all protein coding genes and the gene for snRNA
Pol III: Intermediate sensitivity to α amanitin, transcribes the gene for tRNA, 5S rRNA, U6, RNA component of SRP
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Transcription factorsTranscription factorsDefinition:Definition: Proteins other than purified RNA polymerase subunits, involved in Proteins other than purified RNA polymerase subunits, involved in Initiation, Elongation or Termination of transcriptionInitiation, Elongation or Termination of transcription
Classification:Classification:A: General transcription factors (GTFs):A: General transcription factors (GTFs):
1- are needed for basal gene expression1- are needed for basal gene expression
2- are required for RNA polymerase to bind avidly and specifically 2- are required for RNA polymerase to bind avidly and specifically to the promoters. to the promoters.
3-GTFs for RNA polymerase II are called TFIIA , TFIIB , TFIID , …3-GTFs for RNA polymerase II are called TFIIA , TFIIB , TFIID , …
4- can have multiple subunits4- can have multiple subunits
B: Specific transcription factors:B: Specific transcription factors:
They are used for fine tune control of gene expression of specific They are used for fine tune control of gene expression of specific genesgenes
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RNAP II termination and poly RNAP II termination and poly adenylationadenylation
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mRNA modificationmRNA modificationThe modifications signal the mRNA is ready to move The modifications signal the mRNA is ready to move
out of the nucleus and may control its life span in out of the nucleus and may control its life span in the cytoplasmthe cytoplasm
1) 1) 5’ cap5’ cap:: modified guanine; protection; modified guanine; protection; recognition site for ribosomesrecognition site for ribosomes
2) 2) 3’ tail3’ tail:: poly(A) tail (adenine); protection; poly(A) tail (adenine); protection; recognition; transportrecognition; transport
3) 3) RNA splicingRNA splicing:: exons (expressed sequences) exons (expressed sequences) kept, introns (intervening sequences) spliced out; kept, introns (intervening sequences) spliced out; spliceosomespliceosome
4) 4) Alternative SplicingAlternative Splicing 5) 5) RNA editingRNA editing
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Mechanism of RNA Mechanism of RNA SplicingSplicing
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Alternative splicingAlternative splicing
Calcitonin gene-related peptide
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Toxins and antibiotics that inhibit transcription
Actinomycine D:Actinomycine D: makes cross link makes cross link between the two strands of DNA, between the two strands of DNA, preventing it from being template for preventing it from being template for transcriptiontranscription
Rifamycine ( Rifampin):Rifamycine ( Rifampin): inhibits inhibits beta subunit of RNAP of prokaryotesbeta subunit of RNAP of prokaryotes
Ricin:Ricin: A toxin from plant that inhibits A toxin from plant that inhibits RNAP of eukaryotesRNAP of eukaryotes
Amanitin:Amanitin: A toxin from mashroom A toxin from mashroom that inhibits RNAP of eukaryotesthat inhibits RNAP of eukaryotes