Transcript No Slide Title

Activated transcription by Pol II
enhancers are sequences 5’ to TATAA
transcriptional activators bind them
• have distinct DNA binding and activation domains
Activated transcription by Pol II
enhancers are sequences 5’ to TATAA
transcriptional activators bind them
• have distinct DNA binding and activation domains
• activation domain interacts with mediator
• helps assemble initiation complex on TATAA
Activated transcription by Pol II
enhancers are sequences 5’ to TATAA
transcriptional activators bind them
• have distinct DNA binding and activation domains
• activation domain interacts with mediator
• helps assemble initiation complex on TATAA
•Recently identified “activating RNA”: bind enhancers &
mediator
Activated transcription by Pol II
•Other lncRNA “promote transcriptional poising” in
yeast
http://www.plosbiology.org/article/info%3Adoi%2F10.13
71%2Fjournal.pbio.1001715
•lncRNA displaces
glucose-responsive
repressors & corepressors from genes
for galactose catabolism
•Speeds induction of
GAL genes
Euk gene regulation
Initiating transcription is 1st &
most important control
Most genes are condensed
only express needed genes
not enough room in nucleus to
access all genes at same time!
must find & decompress gene
First “remodel” chromatin:
• some proteins reposition
nucleosomes
• others acetylate histones
• Neutralizes +ve charge
• makes them release DNA
• role of epigenetics
mRNA PROCESSING
Primary transcript is hnRNA
undergoes 3 processing reactions before export to cytosol
All three are coordinated with transcription & affect gene
expression: enzymes piggy-back on POLII
mRNA PROCESSING
Primary transcript is hnRNA
undergoes 3 processing reactions before export to cytosol
1) Capping addition of 7-methyl G to 5’ end
mRNA PROCESSING
Primary transcript is hnRNA
undergoes 3 processing reactions before export to cytosol
1) Capping addition of 7-methyl G to 5’ end
identifies it as mRNA: needed for export & translation
mRNA PROCESSING
Primary transcript is hnRNA
undergoes 3 processing reactions before export to cytosol
1) Capping addition of 7-methyl G to 5’ end
identifies it as mRNA: needed for export & translation
Catalyzed by CEC attached to POLII
mRNA Processing: RNA editing
Two types: C->U and A->I
mRNA Processing: RNA editing
Two types: C->U and A->I
• Plant mito and cp use C -> U
•>300 different editing events have been detected in plant
mitochondria: some create start & stop codons
mRNA Processing: RNA editing
Two types: C->U and A->I
• Plant mito and cp use C -> U
•>300 different editing events have been detected in plant
mitochondria: some create start & stop codons: way to
prevent nucleus from stealing genes!
mRNA Processing: RNA editing
Human intestines edit APOB mRNA C -> U to create a
stop codon @ aa 2153 (APOB48) cf full-length APOB100
• APOB48 lacks the CTD LDL receptor binding site
mRNA Processing: RNA editing
Human intestines edit APOB mRNA C -> U to create a
stop codon @ aa 2153 (APOB48) cf full-length APOB100
• APOB48 lacks the CTD LDL receptor binding site
• Liver makes APOB100 -> correlates with heart disease
mRNA Processing: RNA editing
Two types: C->U and A->I
• Adenosine de-aminases (ADA) are ubiquitously
expressed in mammals
• act on dsRNA & convert A to I (read as G)
mRNA Processing: RNA editing
Two types: C->U and A->I
• Adenosine de-aminases (ADA) are ubiquitously
expressed in mammals
• act on dsRNA & convert A to I (read as G)
• misregulation of A-to-I RNA editing has been implicated
in epilepsy, amyotrophic lateral sclerosis & depression
mRNA Processing: Polyadenylation
Addition of 200- 250 As to end of mRNA
Why bother?
• helps identify as mRNA
• required for translation
• way to measure age of mRNA
->mRNA s with < 200 As have short half-life
mRNA Processing: Polyadenylation
Addition of 200- 250 As to end of mRNA
Why bother?
• helps identify as mRNA
• required for translation
• way to measure age of mRNA
->mRNA s with < 200 As have short half-life
>50% of human mRNAs have alternative polyA sites!
mRNA Processing: Polyadenylation
>50% of human mRNAs have alternative polyA sites!
mRNA Processing: Polyadenylation
>50% of human mRNAs have alternative polyA sites!
• result : different mRNA, can result in altered export,
stability or different proteins
mRNA Processing: Polyadenylation
>50% of human mRNAs have alternative polyA sites!
• result : different mRNA, can result in altered export,
stability or different proteins
• some thalassemias are due to mis-poly A
mRNA Processing: Polyadenylation
some thalassemias are due to mis-poly A
Influenza shuts down nuclear genes by preventing polyAdenylation (viral protein binds CPSF)
mRNA Processing: Polyadenylation
1) CPSF (Cleavage and Polyadenylation Specificity
Factor) binds AAUAAA in hnRNA
mRNA Processing: Polyadenylation
1) CPSF binds AAUAAA in hnRNA
2) CStF (Cleavage Stimulatory Factor) binds G/U rich
sequence 50 bases downstream
CFI, CFII bind in between
Polyadenylation
1) CPSF binds AAUAAA in hnRNA
2) CStF binds; CFI, CFII bind in between
3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to
AAUAAA
mRNA Processing: Polyadenylation
3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to
AAUAAA
4) PAP adds As slowly, CFI, CFII and CPSF fall off
mRNA Processing: Polyadenylation
4) PAP adds As slowly, CFI, CFII and CPSF fall off
5) PABII binds, add
As rapidly until 250
Coordination of mRNA processing
Splicing and polyadenylation factors bind CTD of RNA
Pol II-> mechanism to coordinate the three processes
Capping, Splicing and Polyadenylation
all start before transcription is done!
Export from Nucleus
Occurs through nuclear
pores
anything >
40 kDa needs
exportin
protein
bound
to 5’ cap
Export from Nucleus
In cytoplasm nuclear proteins fall off, new proteins bind
• eIF4E/eIF-4F bind cap
• also new
proteins bind
polyA tail
• mRNA is
ready to be
translated!
Post-transcriptional regulation
1) mRNA processing
2) export from nucleus
3) mRNA degradation
4) mRNA localization
• RNA-binding proteins
link it to cytoskeleton:
bring it to correct site
or store it
4) mRNA localization
• RNA-binding proteins link it to cytoskeleton:bring it to
correct site or store it
•Some RNA (eg Knotted) are transported into neighboring
cells
4) mRNA localization
• RNA-binding proteins link it to cytoskeleton:bring it to
correct site or store it
•Some RNA are transported
into neighboring cells
•Others are transported t/o the
plant in the phloem (SUT1, KN1)
4) mRNA localization
• RNA-binding proteins link it to cytoskeleton:bring it to
correct site or store it
•Some RNA are transported
into neighboring cells
•Others are transported t/o the
plant in the phloem (SUT1, KN1)
•Also some siRNA & miRNA!
4) mRNA localization
• RNA-binding proteins link it to cytoskeleton:bring it to
correct site or store it
•Some RNA are transported
into neighboring cells
•Others are transported t/o the
plant in the phloem (SUT1, KN1)
•Also some siRNA & miRNA!
•siRNA mediate silencing
• Especially of viruses & TE
4) mRNA localization
• RNA-binding proteins link it to cytoskeleton:bring it to
correct site or store it
•Some RNA are transported
into neighboring cells
•Others are transported t/o the
plant in the phloem (SUT1, KN1)
•Also some siRNA & miRNA!
•siRNA mediate silencing
•MiR399 moves to roots to
destroy PHO2 mRNA upon Pi stress
•PHO2 negatively regulates
Pi uptake
Post-transcriptional regulation
RNA in pollen controls first division after fertilization!
Post-transcriptional regulation
RNA in pollen controls first division after fertilization!
Delivery by pollen ensures correct development doesn’t
happen unless egg is fertilized by pollen
Post-transcriptional regulation
4) mRNA localization
• RNA-binding proteins link it to cytoskeleton: bring it to
correct site or store it
• many are stored in P-bodies! More than just an RNAdestruction site
Post-transcriptional regulation
4) mRNA localization
• RNA-binding proteins link it to cytoskeleton: bring it to
correct site or store it
• many are stored in P-bodies! More than just an RNAdestruction site
•Link with initiation of translation
Initiation in Prokaryotes
1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA
Initiation in Prokaryotes
1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA
2) Complex scans down until finds Shine-Dalgarno sequence, 16S
rRNA binds S-D
Initiation in Prokaryotes
1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA
2) Complex scans down until finds Shine-Dalgarno sequence, 16S
rRNA binds S-D
• Next AUG is Start codon, must be w/in 7-13 bases
Initiation in Prokaryotes
1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA
2) Complex scans down until finds Shine-Dalgarno sequence, 16S
rRNA binds S-D
3) IF2-GTP binds tRNAifMet
complex binds start codon
Initiation in Prokaryotes
1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA
2) Complex scans down until finds Shine-Dalgarno sequence, 16S
rRNA binds S-D
3) IF2-GTP binds tRNAifMet
complex binds start codon
4) Large subunit binds
IF2-GTP -> IF2-GDP
tRNAifMet is in P site
IFs fall off
Elongation
1) EF-Tu brings charged
tRNA into A site
Elongation
1) EF-Tu brings charged
tRNA into A site
• anticodon binds
mRNA codon, EF-Tu-GTP > EF-Tu-GDP
Elongation
1) EF-Tu brings charged tRNA into A site
• anticodon binds codon, EF-Tu-GTP -> EF-Tu-GDP
2) ribosome bonds growing peptide on tRNA at P site to a.a. on tRNA
at A site
Elongation
1) EF-Tu brings charged tRNA into A site
• anticodon binds codon, EF-Tu-GTP -> EF-Tu-GDP
2) ribosome bonds growing peptide on tRNA at P site to a.a. on tRNA
at A site
peptidyl transferase is 23S rRNA!
Elongation
3) ribosome translocates one codon
• old tRNA moves to E site & exits
• new tRNA moves to P site
• A site is free for next tRNA
• energy comes from
EF-G-GTP -> EF-G-GDP+ Pi
Wobbling
1st base of anticodon can form unusual pairs with 3rd base of codon
Wobbling
1st base of anticodon can form unusual
pairs with 3rd base of codon
Wobbling
1st base of anticodon can form unusual pairs with 3rd base of codon
• Reduces # tRNAs needed:
• bacteria have 40 or less (bare minimum is 31 + initiator tRNA)
• Eukaryotes have ~ 50
Termination
1) Process repeats until a stop codon is exposed
2) release factor binds nonsense codon
•3 stop codons = 3 RF in prokaryotes (1 RF binds all 3 stop codons
in euk)
Termination
1) Process repeats until a stop codon is exposed
2) release factor binds nonsense codon
• 3 stop codons = 3 RF in prokaryotes (1 RF binds all 3 stop codons
in euk)
3) Releases peptide from tRNA at P site
4) Ribosome falls apart