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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