Transcription

http://www.hhmi.org/biointeractive/media/DNAi_transcription_vo1-lg.mov

From Gene to Protein

Part 2Goal 1- Understand the process of

transcription • How is RNA made?

•How to get from DNA to RNA

Goal 2- Demonstrate how RNA is made • Transcription- Model activity

Try This!Try This!• Molecular Genetics Alphabet

Online Activity

• Crack this code

DNA nucleotide triplet ACG CysCys

Recap: From Gene to Recap: From Gene to Protein Protein Part 1Part 1

What are the two main processes linking gene to protein?

“Cookbook”(genotype)

“Copy of recipe”

“Dish”(phenotype)

The Central Dogma of The Central Dogma of Molecular BiologyMolecular Biology

replication

transcription translation

Why RNA?Why RNA?

Why RNA?Why RNA?

• Not all genes need to be turned on at once. – We can make an RNA transcript of just

ONE GENE– Now we can make the right protein at

the right time in the right location

Why RNA?Why RNA?

In EUKARYOTES…

•DNA cannot leave the nucleus•BUT proteins are built by the ribosomes in the cytosol!•We need a messenger to transfer the genetic code to the ribosomes

mRNAmRNA

• Messenger RNA (mRNA) is a complementary copy of a gene that CAN leave the nucleus

Gaining Access to DNAGaining Access to DNA

4 Phases of 4 Phases of TranscriptionTranscription

1. Initiation2.Elongation3.Termination4.Processing

(Eukaryotes Only)To make a copy you need 3 things:

1.Something to copy – DNA! (cookbook)2.Something to copy with – Nucleotides! (ingredients)3.Something to make the copy – Enzymes! (recipe)

Initiation Initiation (start)(start)

DNA template (the “something” to copy)

Green fluorescent protein

Goal: to express (turn on)

DNA nucleotides

3’ 5’

1. DNA template (the something to copy)

DNA nucleotides

3’ 5’

2. RNA nucleotides (the something to copy with)

3. RNA polymeraseenzyme(the something to make the copy)

3’ 5’

“TA-TA” box

Promoter region Termination sequence

Nucleotides (T-T-A-T-T-T)

Green fluorescent geneTranscription

factorTranscription

factor

Bind/unwind DNA

Transcription unit“Upstream” “Downstream”

InitiationInitiation• RNA polymerase (RNAP) binds to the

double stranded DNA molecule at a promoter sequence (with the help of initiation (transcription) factors)

• It is able to locally unzip DNA with its own built in helicase activity as it constructs an RNA transcript of the DNA

RNA Polymerase II

Enhancers

PromotersPromoters• DNA sequence

upstream of the gene being transcribed

• Determines where RNAP binds and where transcription begins

• Usually rich in Thymine and Adenine (“TATA” box)

ElongationElongation

• One strand of the unzipped DNA acts as a template for RNA synthesis

SG

P SA

P ST

P SC

P SG

P SG

P SA

P SC

P SC

P3´ 5´

SG

PSG

PST

PSC

PSC

PSG

PSA

PST

PSC

P5´ 3´

Template Strand

ElongationElongation

SU

P

SG

PSG

PST

PSC

PSC

PSG

PSA

PST

PSC

P5´ 3´

Template StrandSC

PSG

P SA

P ST

P SC

P SG

P SG

P SA

P SC

P SC

P3´ 5´

SA

PSG

PSC

PSC

PSU

PSG

PSG

P

5´ 3´

Coding Strand

3’

Green fluorescent gene

Transcription factor

Transcription factor

5’

3’5’

Direction of RNA synthesis

5’ 3’

Elongation Elongation (strand)(strand)

ElongationElongation

• mRNA is transcribed in the 5' to 3' direction

• DNA unwinds only in the region of transcription

• After transcription DNA recoils

• Several RNAPs can work on a single gene at once

One more look at Elongation

TerminationTermination• A terminator sequence on the coding

strand tells RNAP when to stop transcribing the mRNA

• RNAP is released and reused and mRNA is released

3’5’

3’5’

Termination (stop)

Transcription unit Termination sequence

messenger RNA (mRNA)

Green fluorescent gene

Translated

Green fluorescent protein

Transcribed (copied)

DNA transcriptionDNA transcription narrated

Transcription VideosTranscription Videos

• mRNA synthesis

• Stages of Transcription

ProcessingProcessing

• In Eukaryotic cells the RNA transcript is called pre-mRNA (or primary RNA) because it must still be modified before it leaves the nucleus

• Why processing?– Remove introns– Protects from degradation in the

cytoplasm

Introns and ExonsIntrons and Exons

Genes contain both coding regions (exons) and non-coding regions (introns)

Introns and ExonsIntrons and Exons

• To produce a final mRNA transcript, introns must be removed

SplicingSplicing

SplicingSplicing

• The Spliceosome is a complex of multiple proteins (snRNPs) and small nuclear RNA (snRNA)

• snRNA binds a specific mRNA sequences at the beginning and end of an intron forming a loop

• The loop is removed and exons are linked

Cap and Tail

S

G

PS

G

PS

U

PS

C

PS

C

PS

G

PS

A

PS

U

PS

C

P

5´ 3´

S

G

PS

G

PS

G

PS

G

P S

A

PS

A

PS

A

PS

A

P S

A

PS

A

PS

A

PS

A

P

cap poly A tail

mRNA transcrip

t

Cap and TailCap and Tail

• To protect RNA from restriction endonucleases in the cytosol, a poly-A tail is added to the 3‘ end of the pre-mRNA

• As an attachment site for the ribosome, a 5‘ cap of modified Gs is added

Try your hand at this:Try your hand at this:

• DNA Sequence:TACGGAGCGTTTCCAACGGTGCATATT

mRNA Sequence:AUGCCUCGCAAAGGUUGCCACGUAU

AA

This code will determine the order of amino acids in a protein…

Try it again, with Try it again, with introns!introns!

• DNA Sequence: INTRONS are red.TACTTGTTATAGTAGTAGGGAGCGCATCTG

AAAAAATCGGCATTTCCACTTCTCGAAACCACCACGGTGCATTCCATT

mRNA Primary Transcript:AUGAACAAUAUCAUCAUCCCUCGCGUAGA

CUUUUUUAGCCGUAAAGGUGAAGAGCUUUGGUGGUGCCACGUAAGGUAA

mRNA Secondary Transcript:AUGAACAAUAUCCGCGUAGACUUUAAAG

GUGAAGAGCUUUGGGUAAGGUAA