transcription. the central dogma dna mrna protein transcription translation gene expression rpe65...

Transcription

the Central Dogma

DNA

mRNA

Protein

transcription

translation

geneexpression

RPE65 gene

RPE65 protein

Transcription Individual DNA regions (genes) copied to mRNA One DNA strand is template Single-stranded RNA produced

template strand

mRNA

template strand

template strand

template strand

Transcription Overview

Un beau jour, je suis allé au marché pour acheter du pain. Il faisait chaud. Alors, j’ai acheté aussi un

limonade.

Il faisait chaud.

mRNA

DNA

transcription

CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC

CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGCGATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG

gene

• What do we call this strand?

Transcription overview

mRNA

DNA

transcription

CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC

CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGCGATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG

template strand

• What enzyme makes RNA?

Transcription overview

mRNA

DNA

transcription – RNA polymerase

CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC

CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGCGATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG

template strand

• What direction is mRNA made?

Transcription overview

mRNA

DNA

transcription – RNA polymerase

CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 3’5’

CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGCGATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG

template strand

• What direction is the template strand read?

Transcription overview

mRNA

DNA

transcription – RNA polymerase

CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 3’5’

CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGCGATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG

• Which strand does the mRNA look like?

5’3’

Transcription overview

mRNA

DNA

transcription – RNA polymerase

CUACGAGGAGGUGAAGCGAUGCCCCGUAGCCGAUAGUAGC 3’5’

CTACGAGGAGGTGAAGCGATGCCCCGTAGCCGATAGTAGCGATGCTCCTCCACTTCGCTACGGGGCATCGGCTATCATCG

• How do we know where to start and stop?

5’3’

Transcription overview

Transcription overview RNA polymerase synthesizes RNA 5′→ 3′ Starts at promoter, ends at terminator

promoter terminator

+1

5′ UTR

coding region

startcodon

stopcodon

“upstream” “downstream”

DNA

transcription

coding region

startcodon

stopcodon

3′ UTR

5′ 3′mRNA

translation

NH3 COOHprotein

• How is the RNA actually made?

Prokaryotic transcriptionPromoter: -10 and -35 sequences

DNA

-35-35 -10-10 +1+1mRNA

5′ TTGACAT AACTGTA

5′ TATAAT ATATTA

5’3’

Prokaryotic transcriptionPromoter: -10 and -35 sequences

DNA

-35-35 -10-10 +1+1mRNA

TTGACAT TATAAT

5’3’

Prokaryotic transcriptionInitiation:

RNAP sigma subunit (σ) binds -10 and -35

DNA

-35-35 -10-10 +1+1σ5’

3’

Prokaryotic transcriptionInitiation:

RNAP core (α2ββ’) binds sigma

DNA

-35-35 -10-10 +1+1σ

α2 ββ’“core”5’

3’

Prokaryotic transcriptionInitiation:

Promoter determines template strand and direction

-35-3555′′ 33′′33′′ 55′′

-10-10

-35 -35-10 -10

template strandfor gene 1

template strandfor gene 2

Regulatory elements Prokaryotes use operator sequences

DNA

-35-35 -10-10 +1+1mRNA

TTGACAT TATAAT

5’3’

OperatorsProtein Transcription factors

Prokaryotic transcriptionInitiation:

RNAP opens transcription bubble (helicase activity)

DNA

-35-35 -10-10σ

+1+1

5’3’

Prokaryotic transcriptionInitiation:

RNAP begins mRNA synthesis at +1

DNA

-35-35 -10-10σ

+1+1

5’3’ mRNA

Prokaryotic transcriptionInitiation:

Sigma released

DNA

-35-35 -10-10σ

+1+1

5’3’ mRNA

Elongation:

Prokaryotic transcription

DNA

-35-35 -10-10

5’3’

Elongation:

Prokaryotic transcription

DNA5’3’

terminator

Replication Transcription Synthesize DNA Copy whole genome Copy both strands Need primer 5′ → 3′ Multiple enzymes

• How are replication and transcription similar?

• How are they different?

Synthesize RNA Copy one gene Copy one strand No primer 5′ → 3′ Only RNA polymerase

Eukaryotic transcription 3 RNA polymerases:

RNA polymerase I – rRNA RNA polymerase II – mRNA RNA polymerase III – tRNA

RNA polymerase IIfrom yeast

Eukaryotic transcription RNAP II recognizes:

TFIID bound to TATA box (TATAAA) TFIIB bound to TFIID Transcription factors bound to enhancer sequences

+1

Enhancers

Transcription factors

Sp1 hERR1 CAAT GATA TATAbox

TFIIB TFIID

Eukaryotic transcription RNAP II recognizes:

TFIID bound to TATA box (TATAAA) TFIIB bound to TFIID Transcription factors bound to enhancer sequences

+1

Different from Prokaryotes- No terminator! RNA cleaved from transcription complex

+1

AAUAAA

AAUAAA

Eukaryotic Transcription Termination

RNA processing in eukaryotes

DNA

promoter

exons introns primary transcript(nucleus)

5’ capAAAAAAAAA3’ poly-A tail

AAAAAAAAA

splicingsplicing

transcriptiontranscription

unbroken coding sequence

transport to cytoplasm for translationtransport to cytoplasm for translation

final mRNA

methylated guanine “backward” 5′ to 5′ linkage Not encoded in DNA Capping enzyme Recognition by ribosome

5′ cap

5′ AGACCUGACCAUACC

RNA processing in eukaryotes

DNA

promoter

exons introns primary transcript(nucleus)

5’ capAAAAAAAAA3’ poly-A tail

AAAAAAAAA

splicingsplicing

transcriptiontranscription

unbroken coding sequence

transport to cytoplasm for translationtransport to cytoplasm for translation

final mRNA

3′ poly(A) tail Poly(A) polymerase Add ~200 A’s Not in template Important for:

Export of mRNA Initiation of Translation Stability of mRNA

…UGGCAGACCUGACCA 3′

…UGGCAGACCUGACCAAAAAAAAAAAAAAAAAAAA

RNA processing in eukaryotes

DNA

promoter

exons introns primary transcript(nucleus)

5’ capAAAAAAAAA3’ poly-A tail

AAAAAAAAA

splicingsplicing

transcriptiontranscription

unbroken coding sequence

transport to cytoplasm for translationtransport to cytoplasm for translation

final mRNA

Splicing Most genes interrupted by introns Introns removed after transcription Exons spliced together

5’ capAAAAAAAAA3’ poly-A tail

AAAAAAAAA

splicingsplicing

unbroken coding sequencefinal mRNA

Splicing snRNPs recognize exon-intron

boundaries RNA + protein Cut and rejoin mRNA

Splicing

RPE65 mRNA in nucleus: 21,000 nt (14 exons)

AAAAAAAAA

AAAAAAAAA

splicingsplicing

mature RPE65 mRNA in nucleus: 1,700 nt (8%)

Splicing Alternative splicing: >1 protein from one gene 27,000 human genes, but >100,000 proteins

Splicing

Mutations affecting splicing can cause genetic disease:cystic fibrosis retinitis pigmentosaspinal muscular atrophy Prader-Willi syndromeHuntington disease spinocerebellar ataxiamyotonic dystrophy Fragile-X syndrome

Or produce genetic susceptibility to disease:lupus bipolar disorderschizophrenia myocardial infarctiontype I diabetes asthmacardiac hypertrophy multiple sclerosisautoimmune diseases elevated cholesterol