1. Activated transcription by Pol II
enhancers are sequences 5’ to TATAA
transcriptional activators bind them
have distinct DNA binding and activation domains

2. 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

3. 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

4. Activated transcription by Pol II
Other lncRNA “promote transcriptional poising” in yeast
lncRNA displaces
glucose-responsive
repressors & co-
repressors from genes
for galactose catabolism
Speeds induction of
GAL genes

5. 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

6. First “remodel” chromatin:
some proteins reposition
nucleosomes
others acetylate histones
Neutralizes +ve charge
makes them release DNA
role of epigenetics

7. 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

8. mRNA PROCESSING
Primary transcript is hnRNA
undergoes 3 processing reactions before export to cytosol
1) Capping addition of 7-methyl G to 5’ end

9. 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

10. 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

11. mRNA Processing: RNA editing
Two types: C->U and A->I

12. 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

13. 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!

14. mRNA Processing: RNA editing
Human intestines edit APOB mRNA C -> U to create a stop aa 2153 (APOB48) cf full-length APOB100
APOB48 lacks the CTD LDL receptor binding site

15. mRNA Processing: RNA editing
Human intestines edit APOB mRNA C -> U to create a stop aa 2153 (APOB48) cf full-length APOB100
APOB48 lacks the CTD LDL receptor binding site
Liver makes APOB100 -> correlates with heart disease

16. 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)

17. 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

18. mRNA Processing: Polyadenylation
Addition of 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

19. mRNA Processing: Polyadenylation
Addition of 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!

20. mRNA Processing: Polyadenylation
>50% of human mRNAs have alternative polyA sites!

21. mRNA Processing: Polyadenylation
>50% of human mRNAs have alternative polyA sites!
result : different mRNA, can result in altered export, stability or different proteins

22. 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

23. mRNA Processing: Polyadenylation
some thalassemias are due to mis-poly A
Influenza shuts down nuclear genes by preventing poly-Adenylation (viral protein binds CPSF)

24. mRNA Processing: Polyadenylation
1) CPSF (Cleavage and Polyadenylation Specificity Factor) binds AAUAAA in hnRNA

25. 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

26. Polyadenylation
1) CPSF binds AAUAAA in hnRNA
2) CStF binds; CFI, CFII bind in between
3) PAP (PolyA polymerase) binds & cleaves b 3’ to AAUAAA

27. mRNA Processing: Polyadenylation
3) PAP (PolyA polymerase) binds & cleaves b 3’ to AAUAAA
4) PAP adds As slowly, CFI, CFII and CPSF fall off

28. mRNA Processing: Polyadenylation
4) PAP adds As slowly, CFI, CFII and CPSF fall off
PABII binds, add
As rapidly until 250

29. 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!

30. Export from Nucleus
Occurs through nuclear
pores
anything >
40 kDa needs
exportin
protein
bound
to 5’ cap

31. 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!

32. 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

33. 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

34. 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)

35. 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!

36. 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

37. 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

38. Post-transcriptional regulation
RNA in pollen controls first division after fertilization!

39. 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

40. 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 RNA-destruction site

41. 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 RNA-destruction site
Link with initiation of translation

42. Initiation in Prokaryotes
1) IF1 & IF3 bind 30S subunit, complex binds 5' mRNA

43. Initiation in Prokaryotes
IF1 & IF3 bind 30S subunit, complex binds 5' mRNA
Complex scans down until finds Shine-Dalgarno sequence, 16S rRNA binds S-D

44. Initiation in Prokaryotes
IF1 & IF3 bind 30S subunit, complex binds 5' mRNA
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

45. Initiation in Prokaryotes
IF1 & IF3 bind 30S subunit, complex binds 5' mRNA
Complex scans down until finds Shine-Dalgarno sequence, 16S rRNA binds S-D
IF2-GTP binds tRNAifMet
complex binds start codon

46. Initiation in Prokaryotes
IF1 & IF3 bind 30S subunit, complex binds 5' mRNA
Complex scans down until finds Shine-Dalgarno sequence, 16S rRNA binds S-D
IF2-GTP binds tRNAifMet
complex binds start codon
Large subunit binds
IF2-GTP -> IF2-GDP
tRNAifMet is in P site
IFs fall off

47. Elongation
1) EF-Tu brings charged tRNA into A site

48. Elongation
EF-Tu brings charged tRNA into A site
anticodon binds
mRNA codon, EF-Tu-GTP -> EF-Tu-GDP

49. Elongation 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

50. Elongation 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!

51. 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

52. Wobbling
1st base of anticodon can form unusual pairs with 3rd base of codon

53. Wobbling
1st base of anticodon can form unusual
pairs with 3rd base of codon

54. 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

55. 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)

56. 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