1. Regulating transcription
Telling RNA pol to copy a DNA sequence
Transcription factors bind promoters & control initiation of transcription
1/signal gene senses
1 binding site/signal gene senses

2. Transcription factors
Bind surface -> base-pairs form unique patterns in major & minor grooves

3. Transcription factors
Bind surface -> base-pairs form unique patterns in major & minor grooves
Scan DNA for correct pattern
need H-bonds
= 5-8 base-pairs

4. Transcription
Prokaryotes have one RNA polymerase
makes all RNA
core polymerase = complex of 5 subunits (a1aIIbb’w)
w not absolutely needed, but cells lacking w are very sick

5. Initiating transcription in Prokaryotes
1) Core RNA polymerase is promiscuous

6. Initiating transcription in Prokaryotes
Core RNA polymerase is promiscuous
sigma factors provide specificity
Bind promoters

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

8. Initiating transcription in Prokaryotes
Core RNA polymerase is promiscuous
sigma factors provide specificity
Bind promoters
3) Once bound, RNA polymerase
“melts” the DNA

9. Initiating transcription in Prokaryotes
3) Once bound, RNA polymerase
“melts” the DNA
4) rNTPs bind template

10. 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

11. 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

12. Structure of Prokaryotic promoters
Three DNA sequences (core regions)
1) Pribnow box at -10 (10 bp 5’ to transcription start)
5’-TATAAT-3’ determines exact start site: bound by s factor

13. Structure of Prokaryotic promoters
Three DNA sequences (core regions)
1) Pribnow box at -10 (10 bp 5’ to transcription start)
5’-TATAAT-3’ determines exact start site: bound by s factor
2)” -35 region” : 5’-TTGACA-3’ : bound by s factor

14. Structure of Prokaryotic promoters
Three DNA sequences (core regions)
1) Pribnow box at -10 (10 bp 5’ to transcription start)
5’-TATAAT-3’ determines exact start site: bound by s factor
2)” -35 region” : 5’-TTGACA-3’ : bound by s factor
3) UP element : -57: bound by a factor

15. Structure of Prokaryotic promoters
Three DNA sequences (core regions)
1) Pribnow box at -10 (10 bp 5’ to transcription start)
5’-TATAAT-3’ determines exact start site: bound by s factor
2)” -35 region” : 5’-TTGACA-3’ : bound by s factor
3) UP element : -57: bound by a factor

16. Structure of Prokaryotic promoters
Three DNA sequences (core regions)
1) Pribnow box at -10 (10 bp 5’ to transcription start)
5’-TATAAT-3’ determines exact start site: bound by s factor
2)” -35 region” : 5’-TTGACA-3’ : bound by s factor
3) UP element : -57: bound by a factor
Other sequences also often influence transcription! Eg Trp operator

17. Prok gene regulation
5 genes (trp operon) encode trp enzymes

18. Prok gene regulation
Copy genes when no trp
Repressor stops operon if [trp]

19. Prok gene regulation
Repressor stops operon if [trp]
trp allosterically regulates repressor
can't bind operator until 2 trp bind

20. lac operon
Some operons use combined “on” & “off” switches E.g. E. coli lac operon
Encodes enzymes to use lactose
lac Z = -galactosidase
lac Y= lactose permease
lac A = transacetylase

21. lac operon
Make these enzymes only if:
1) - glucose

22. lac operon
Make these enzymes only if:
1) - glucose
2) + lactose

23. lac operon
Regulated by 2 proteins
1) CAP protein : senses [glucose]

24. lac operon
Regulated by 2 proteins
CAP protein : senses [glucose]
lac repressor: senses [lactose]

25. lac operon
Regulated by 2 proteins
CAP protein : senses [glucose]
lac repressor: senses [lactose]
encoded by lac i gene
Always on

26. lac operon
2 proteins = 2 binding sites
1) CAP site: promoter isn’t active until CAP binds

27. lac operon
2 proteins = 2 binding sites
CAP site: promoter isn’t active until CAP binds
Operator: repressor blocks transcription

28. lac operon
Regulated by 2 proteins
1) CAP only binds if no glucose
-> no activation

29. lac operon
Regulated by 2 proteins
1) CAP only binds if no glucose
-> no activation
2) Repressor blocks transcription if no lactose

30. lac operon
Regulated by 2 proteins
1) CAP only binds if no glucose
2) Repressor blocks transcription if no lactose
3) Result: only make enzymes for using lactose if lactose is present and glucose is not

32. Result
[-galactosidase]
rapidly rises if no
glucose & lactose
is present
W/in 10 minutes
is 6% of total
protein!

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

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

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

36. 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

37. 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

38. 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

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

40. 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”

41. 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

42. 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

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

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

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

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

47. 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

48. 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

49. 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

50. 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

51. 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)

52. 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

53. 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

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

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

56. 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+

57. 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]

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

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

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

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

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

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

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

65. 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

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

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

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

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

70. 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

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

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

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

74. 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

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

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

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

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

79. 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

80. 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

81. 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

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

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

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

86. 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

87. 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

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

89. 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