1. Regulating gene expression
Goal is
controlling
Proteins
How many?
Where?
How active?
8 levels (two not
shown are mRNA
localization & prot
degradation)

2. Regulating gene expression
1) initiating
transcription
most important
~50% of control

3. Initiating transcription in Prokaryotes
Core RNA polymerase is promiscuous
sigma factors provide specificity
Bind promoters
Different sigmas bind different promoters

4. Initiating transcription in Prokaryotes
3) Once bound, RNA polymerase
“melts” the DNA
4) rNTPs bind template
5) RNA polymerase catalyzes phosphodiester bonds, melts and unwinds template
6) sigma falls off after ~10 bases are added

6. Structure of Prokaryotic promoters
Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter.

7. Structure of Prokaryotic promoters
Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter. ↵

8. Structure of Prokaryotic promoters
Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter.
nrsBACD encode nickel transporters

9. Structure of Prokaryotic promoters
Other sequences also often influence transcription! Bio502 plasmid contains the nickel promoter.
nrsBACD encode nickel transporters
nrsRS encode “two component” signal transducers
nrsS encodes a his kinase
nrsR encodes a response regulator

10. Structure of Prokaryotic promoters
nrsRS encode “two component” signal transducers
nrsS encodes a his kinase
nrsR encodes a response regulator
When nrsS binds Ni it kinases nrsR

11. Structure of Prokaryotic promoters
nrsRS encode “two component” signal transducers
nrsS encodes a his kinase
nrsR encodes a response regulator
When nrsS binds Ni it kinases nrsR
nrsR binds Ni promoter and activates
transcription of both operons

12. Termination of transcription in prokaryotes
1) Sometimes go until ribosomes fall too far behind

13. Termination of transcription in prokaryotes
1) Sometimes go until ribosomes fall too far behind
2) ~50% of E.coli genes require a termination factor called “rho”

14. Termination of transcription in prokaryotes
1) Sometimes go until ribosomes fall too far behind
2) ~50% of E.coli genes require a termination factor called “rho”
3) rrnB first forms an RNA hairpin, followed by an 8 base sequence TATCTGTT that halts transcription

15. Transcription in Eukaryotes
3 RNA polymerases
all are multi-subunit
complexes
5 in common
3 very similar
variable # unique ones
Now have Pols IV & V in plants
Make siRNA

16. Transcription in Eukaryotes
RNA polymerase I: 13 subunits ( unique)
acts exclusively in nucleolus to make 45S-rRNA precursor

17. Transcription in Eukaryotes
Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor
accounts for 50% of total RNA synthesis

18. Transcription in Eukaryotes
Pol I: acts exclusively in nucleolus to make 45S-rRNA precursor
accounts for 50% of total RNA synthesis
insensitive to -aminitin

19. Transcription in Eukaryotes
Pol I: only makes 45S-rRNA precursor
50 % of total RNA synthesis
insensitive to -aminitin
Mg2+ cofactor
initiation frequency

20. promoter is 5' to "coding sequence" 2 elements
RNA polymerase I
promoter is 5' to "coding sequence"
2 elements
1) essential core includes transcription start site +1
-100
coding sequence
UCE
core

21. promoter is 5' to "coding sequence" 2 elements
RNA polymerase I
promoter is 5' to "coding sequence"
2 elements
1) essential core includes transcription start site
2) UCE (Upstream Control Element) at ~ stimulates transcription x +1
-100
coding sequence
UCE
core

22. Initiation of transcription by Pol I
Order of events was determined by in vitro reconstitution
1) UBF (upstream binding factor) binds UCE and core element
UBF is a transcription factor: DNA-binding proteins which recruit polymerases and tell them where to begin

23. Initiation of transcription by Pol I
1) UBF binds UCE and core element
2) SL1 (selectivity factor 1) binds UBF (not DNA)
SL1 is a coactivator
proteins which
bind transcription
factors and
stimulate
transcription

24. Initiation of transcription by Pol I
1) UBF binds UCE and core element
2) SL1 (selectivity factor 1) binds UBF (not DNA)
SL1 is a complex of
4 proteins including
TBP (TATAA-
binding protein)

25. Initiation of transcription by Pol I
1) UBF binds UCE and core element
2) SL1 (selectivity factor 1) binds UBF (not DNA)
3) complex recruits Pol I

26. Initiation of transcription by Pol I
1) UBF binds UCE and core element
2) SL1 (selectivity factor 1) binds UBF (not DNA)
3) complex recruits Pol I
4) Pol I transcribes until
it hits a termination site

27. Processing rRNA
~ 200 bases are methylated
Box C/D snoRNA picks sites

28. Processing rRNA
~ 200 bases are methylated
Box C/D snoRNA picks sites
One for each!

29. Processing rRNA
~ 200 bases are methylated
Box C/D snoRNA picks sites
One for each!
2) Another ~ 200 are pseudo-uridylated
Box H/ACA snoRNAs pick sites

30. Processing rRNA
~ 400 bases are methylated or pseudo-uridylated
snoRNAs pick sites
One for each!
2) 45S pre-rRNA is cut into 28S, 18S and 5.8S products

31. Processing rRNA
~ 200 bases are methylated
2) 45S pre-rRNA is cut into 28S,
18S and 5.8S products
3) Ribosomes are assembled w/in nucleolus

32. RNA Polymerase III
Reconstituted in vitro
makes ribosomal 5S and tRNA
(+ some snRNA & scRNA)

33. RNA Polymerase III
makes ribosomal 5S and tRNA
(+ some snRNA & scRNA)
>100 different kinds of genes
~10% of all RNA synthesis

34. RNA Polymerase III
makes ribosomal 5S and tRNA
(+ some snRNA & scRNA)
>100 different kinds of genes
~10% of all RNA synthesis
Cofactor = Mn2+ cf Mg2+

35. RNA Polymerase III
makes ribosomal 5S and tRNA
(+ some snRNA & scRNA)
>100 different kinds of genes
~10% of all RNA synthesis
Cofactor = Mn2+ cf Mg2+
sensitive to high [-aminitin]

36. RNA Polymerase III
makes ribosomal 5S and tRNA (also some snRNA and some scRNA)
Has the most subunits

37. RNA Polymerase III
makes ribosomal 5S and tRNA (also some snRNA and some scRNA)
Has the most subunits
initiation frequency

38. Initiation of transcription by Pol III
promoter is often w/in "coding" sequence!

39. Initiation of transcription by Pol III
promoter is w/in "coding" sequence!
5S & tRNA promoters differ
5S has single “C” box

40. Initiation of transcription by Pol III
1) TFIIIA binds C box in 5S
2) recruits TFIIIC

41. Initiation of transcription by Pol III
TFIIIA binds C box
recruits TFIIIC
3) TFIIIB binds
TBP & 2 others

42. Initiation of Pol III transcription
1) TFIIIA binds C box
2) recruits TFIIIC
3) TFIIIB binds
4) Complex recruits Pol III

43. Initiation of Pol III transcription
1) TFIIIA binds C box
2) recruits TFIIIC
3) TFIIIB binds
4) Complex recruits Pol III
5) Pol III goes until
hits > 4 T's

44. Initiation of transcription by Pol III
promoter is w/in coding sequence!
5S & tRNA promoters differ
tRNA genes have “A” & “B” boxes

45. Initiation of transcription by Pol III
tRNA genes have “A” and “B” boxes
1) TFIIIC binds B box

46. Initiation of transcription by Pol III
tRNA genes have “A” and “B” boxes
1) TFIIIC binds B box
2) recruits TFIIIB

47. Initiation of transcription by Pol III
1) TFIIIC binds box B
2) recruits TFIIIB
3) complex recruits Pol III

48. Initiation of transcription by Pol III
1) TFIIIC binds box B
2) recruits TFIIIB
3) complex recruits Pol III
4) Pol III runs until
hits > 4 Ts

49. Processing tRNA
tRNA is trimmed
5’ end by RNAse P
(contains RNA)
3’ end by RNAse Z
Or by exonucleases

50. Processing tRNA
tRNA is trimmed
Transcript is spliced
Some tRNAs are
assembled from 2 transcripts

51. transferase (no template)
Processing tRNA
tRNA is trimmed
Transcript is spliced
CCA is added to 3’ end
By tRNA nucleotidyl
transferase (no template)
tRNA +CTP -> tRNA-C + PPi tRNA-C +CTP--> tRNA-C-C + PPi tRNA-C-C +ATP -> tRNA-C-C-A + PPi

52. Processing tRNA
tRNA is trimmed
Transcript is spliced
CCA is added to 3’ end
Many bases are modified
Significance unclear

53. Processing tRNA
tRNA is trimmed
Transcript is spliced
CCA is added to 3’ end
Many bases are modified
No cap! -> 5’ P
(due to 5’ RNAse P cut)

54. RNA Polymerase II
makes mRNA (actually hnRNA), some snRNA and scRNA

55. RNA Polymerase II
makes mRNA (actually hnRNA), some snRNA and scRNA
>30,000 different genes

56. RNA Polymerase II
makes mRNA (actually hnRNA), some snRNA and scRNA
>30,000 different genes
20-40% of total RNA synthesis

57. RNA Polymerase II
makes mRNA (actually hnRNA), some snRNA and scRNA
~ 30,000 different genes
20-40% of all RNA synthesis
very sensitive to -aminitin

58. RNA Polymerase II
12 subunits in yeast,
unknown elsewhere

59. RNA Polymerase II
12 subunits in yeast,
unknown elsewhere
Largest subunit (L’) has
CarboxyTerminal
Domain (CTD)
important role in
regulating pol II

60. Initiation of transcription by Pol II
Needs > 30 other factors to initiate transcription
final complex is called a transcriptosome
contains > 50 proteins

61. Initiation of transcription by Pol II
Separate basal and activated transcription
basal transcription is not regulated
driven by minimal promoter

62. Initiation of transcription by Pol II
Separate basal and activated transcription
basal transcription is not regulated
driven by minimal promoter
TATAA box at -30
TATAA
-30 +1
coding sequence

63. Initiation of transcription by Pol II
Separate basal and activated transcription
activated transcription is regulated by proteins bound to promoter elements called enhancers and silencers
usually 5’ to TATAA box
TATAA
-30 +1
coding sequence
UCE

64. Initiation of transcription by Pol II
Separate basal and activated transcription
activated transcription is regulated by proteins bound to promoter elements called enhancers and silencers
usually 5’ to TATAA box
Requires nucleosome repositioning
TATAA
-30 +1
coding sequence
UCE

66. Initiation of transcription by Pol II
Basal transcription
1) TFIID (includingTBP) binds TATAA box

67. Initiation of transcription
by Pol II
Basal transcription
1) TFIID binds to
TATAA box
2) Distorts DNA

68. Initiation of transcription by Pol II
Basal transcription
1) TFIID binds TATAA box
2) TFIIA and TFIIB bind TFIID/DNA

69. Initiation of transcription by Pol II
Basal transcription
1) TFIID binds TATAA box
2) TFIIA and TFIIB bind
TFIID/DNA
3) Complex recruits Pol II

70. Initiation of transcription by Pol II
Basal transcription
1) TFIID binds TATAA box
2) TFIIA and TFIIB bind to
TFIID/DNA
3) Complex recruits Pol II
4) Still must recruit
TFIIE & TFIIH to
form initiation complex

71. Initiation of transcription by Pol II
Basal transcription
1) Once assemble initiation complex must start Pol II
2) TFIIH kinases CTD

72. Initiation of transcription by Pol II
Basal transcription
1) Once assemble initiation complex must start Pol II
2) TFIIH kinases CTD
negative charge
gets it started
3) Exchange initiation
for elongation factors

73. Initiation of transcription by Pol II
Basal transcription
1) Once assemble initiation complex must start Pol II
2) Kinase CTD
negative charge
gets it started
3) Exchange initiation
for elongation factors
4) Continues until
hits terminator

74. Initiation of transcription by Pol II
Basal transcription
1) Once assemble initiation complex must start Pol II
2) Kinase CTD
negative charge
gets it started
3) RNA pol II is paused
on many promoters!

75. Initiation of transcription by Pol II
Basal transcription
1) Once assemble initiation complex must start Pol II
2) Kinase CTD
negative charge
gets it started
3) RNA pol II is paused
on many promoters!
even of genes that
aren’t expressed!
(low [mRNA])

76. Initiation of transcription by Pol II
RNA pol II is paused on many promoters!
even of genes that aren’t expressed! (low [mRNA])
Early elongation is also
regulated!