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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