Transcription in bacteria

1. Transcription in bacteria

MSU & SkolTech
Transcription
in bacteria
1755

2. Transcription

RNA synthesis
5'
...O
O
B1 '
B1
DNA
O OH
O P O
O
-
RNA
5'
...O
B2
O
O P O
O
-
O
O
-
O
B2
O
-
B2 '
O
DNA
O OH
O
O P O P O P O
-
B1'
O OH
B2 '
OH OH
3'
O
B1
-
O
B3
O P O
B3 '
-
O
B3
B3 '
O
O
RNA
OHOH
O
O
-
OH OH
3'
-
O P O P O
-
O
-
O
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3. Transcription

RNA bases
N
H2N
N
O
O
NH
N
N
N
O
N
C
N
G
N
A
NH
NH2
N
N
H2N
O
U
1755

4. Transcription

RNA polymerase
a2bb’ws
Core
a 40 kD
b 155 kD
b’ 165 kD
w 10 kD
RNA polymerase
s 70 major
s 20 Fe2+ transport
s 28 flagella
s 32 heat shock
s 54 nitrogen limitation
s 24 periplasm stress
s 38 stationary phase
1755

5. Transcription

Proteins that associate with RNAP during elongation
NusA
55kD pausing, antitermination
NusG
21kD
GreA
suppression of pausing,
antitermination
18kD cleavage of 2-3 n. in
stalled RNAP elongation
complexes
GreB
19kD cleavage of 9 n. in stalled
RNAP elongation complexes
Mfd
130kD release of RNAP
blocked
by DNA lesions,
activation of UvrABC
Termination
factor
r 47 kD, hexamer,
ATPase
1755

6. Transcription

Initiation of transcription
-10
-35
TTGACA
s
s
a2bb’
a2bb’
promoter
TATAAT
s
a2bb’
a2bb’
1755

7. Transcription

Promoters and s factors
-35
-10
E.coli s70
TTGACA
TATAAT
E.coli s32
TCTC-CCCTTGAA
CCCCAT-TA
E.coli s54
(-24)CTGG-A
(-12)TTGCA
B. sub sA
TTGACA
TATAAT
B. sub sB
AGGTTTAA
GGGTAT
B. sub sD
CTAAA
CCGATAT
B. sub sE
ATATT
ATACA
B. sub sK
AC
CATA---T
B. sub sH
CAGGA
GAATT—T
SPO1sgp28
AGGAGA
TTT-TTT
T4sgp55
-
TATAAATA
s
1755

8. Transcription

Structure of initiation complex
s4
s3
TTGACA
TG
-35
s2
s1
TATAAT GGGA
+1
-10
Core
Core
σ2
TG -10
GGGA
σ1
σ4
-35
Mg2+
1755

9. Transcription

Structure of initiation complex
s4
s3
TTGACA
TG
-35
s2
s1
TATAAT GGGA
+1
-10
Core
Core
σ2
TG -10
GGGA
σ1
RNA
Mg2+
1755

10. Transcription

Elongation
NusG
NusA
NusG
a2bb’
NusA
a2bb’
Pausing, regulation
or secondary structure
formation
Elongation
NusG
NusG
NusA
a2bb’
GreA/B
NusA
a2bb’
Mfd
TT
Release of stalled
complexes
Release of RNAP
stalled at DNA lesions
1755

11. Transcription

GreA use the tunnel to access RNAP active center
b’
RNAP
w
GreA
a
b
NTP
tunnel
a
1755

12. Transcription

Termination
r-dependent
NTP
NDP
NusA
r
loading
Pause,
termination
NusG
a2bb’
r-independent
UUUU
a2bb’
1755

13. Transcription

r-factor
1755

14. Regulation of transcription

Regulation by s-factor change
O
O
O
O
2NH3+2[H]
HO
OH
H2N
OH
O
NH2
low nitrogen
UTase/UR
UTase/UR
excess nitrogen
PII
PII
PII
PII
UMP
NtrB
NtrB PII
PO4
NtrC
UMP
PO4
NtrC
NtrC
NtrC
PO4
NtrC
s54
RNAP
1755

15. Regulation of transcription

DNA looping
1755

16. Regulation of transcription

Regulation of stationary s-factor expression
5'
AUG
SD
5'
RpoS ORF
3'
Dsr A RNA
5'
5'
translation
AUG
SD
RpoS ORF
1755

17. Regulation of transcription

Regulation by DNA binding regulator proteins
lac operon:
Glucose is preferable
energy source
Lactose could be utilized
if available
Jacob, Monod
Nobel Prize
1755

18. Regulation of transcription

Regulation by DNA binding regulator proteins
No glucose
No lactose
Glucose
No lactose
O O
CRP
O O
LacI LacI
LacI LacI
P
P
No glucose
Lactose
Glucose
Lactose
P
O
LacZ
O
CRP
RNAP
P
O
LacZ
O
1755

19. Regulation of transcription

Regulation by DNA binding regulator proteins
How activation works?
aCTD
Activator
s70
aCTD
}
-35
a
a
b’
b
Activator
aCTD
s70
aCTD
-10
a
a
-35
b’
b
-10
24-54 bp
Activation via a-subunit CTD
1755

20. Regulation of transcription

Regulation by DNA binding regulator proteins
How activation works?
s70
a
a
-10
-35
MerR
b’
b
s70
-35 2+
Hg
a
a
b’
b
-10
2+
Hg
MerR
Activation/repression via DNA structure change
1755

21. Regulation of transcription

Regulation by DNA binding regulator proteins
Complex regulation
Nir-operon
Fis
No oxygen
IHF
FNR FNR
-35
Nitrite ions present
-10
No oxygen
IHF
aCTD
Fis
NarL/NarP
FNR
-35
a
a
s70
b’
b
-10
1755

22. Regulation of transcription

Regulation by DNA binding regulator proteins
Transcription factor activity change by localization
MBP
MalG
MBP
MalF
MalK MalK
ATP
ATP
MalT
MalG
MalK
ADP
MalT
MalF
MalK
ADP
transcription
1755

23. Regulation of transcription

Regulation by low molecular weight compounds
Low UTP
5’ AAUUUG
mRNA
RNAP
Excess UTP: abortive
transcription
5’
AAUUUUUU
5’
AAUUUUU
RNAP
1755

24. Regulation of transcription

Regulation by low molecular weight compounds
ppGpp
DksA
GreA
O
N
O
-
O
O
P O P O
-
-
O
N
O
O
-
O
-
O
O
NH
NH 2
OH
-
P O P O
O
O
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25. Regulation of transcription

Regulation of elongation and termination
Attenuation of E. coli Trp operon
Low Trp
concentration
ribosome
mRNA
5’
1
2
3
a2bb’
4
UGG UGG
High Trp
concentration
ribosome
3
mRNA
5’
1
UGG UGG
2
4
UUUUUUUUUUU
1755

26. Regulation of transcription

Regulation of elongation and termination
Attenuation of E. coli pyrimidine operon
Low UTP
concentration
ribosome
mRNA
5’
UUUUU
1
2
RNAP
High UTP
concentration
ribosome
mRNA
5’
1
2
UUUUUUUUUUU
1755

27. Regulation of transcription

Regulation of elongation and termination
Attenuation of B. subtilis Trp operon
Low Trp
concentration
1
2
3
5’
TRAP
RNAP
High Trp
concentration
Trp
5’
TRAP
3
2
UUUUU 3’
1
1755

28. Regulation of transcription

Regulation of elongation and termination
Attenuation of B. subtilis TyrRS gene
transcription
termination
of Tyr-ARS
UUUUUU
Tyr-тРНК
Tyr
High
aminoacylation
тРНК
Tyr
Low
aminoacylation
1755

29. Regulation of transcription

Regulation of elongation and termination
xpt mRNA
B. subtilis
repR mRNA
antisense RNA III
UUUUUU
termination
UUUUUU
no RNA III
transcription
repR
high concentration of
guanine
low concentration of
guanine
1755

30. Regulation of transcription

Regulation of elongation and termination
s
-35
-10
Q
a2bb’
Antitermination Q
ter
pause
qut
NusG
S10
N
Antitermination N
boxA
nut
NusA
boxB
a2bb’
1755

31. Regulation of transcription

Regulation of initiation
nucleotides
activators
s
a2bb’
alternative
-factors
s
repressor
enhancer
1755

32. Regulation of transcription

Regulation of elongation and termination
Attenuation
1
2
3
N
NusG
a
bb
’
2
NusA
a2bb’
- translation speed
-transcription speed
-protein binding
-RNA binduing
-small molecule binding
Antitermination
- RNA binding protein
antiterminator
- DNA binding protein
antiterminator
1755

33. Examples of the transcriptional control

Bacterial flagella biosynthesis
Flagella is expensive and energy consuming machinery
1755

34. Examples of the transcriptional control

Bacterial flagella biosynthesis
low glucose
H-NS
cAMP
quorum sensing
AI-2
QseC
CRP
P
QseB
FlhD
P
HdfR
OmpR
EnvZ
DnaKJ,GrpE?
high
osmolarity
FlhC
high temperature
?
LrhA
RscC
? ?
RscD
P
RscA
RscB
P
Many regulators control flagella biogenesis
1755

35. Examples of the transcriptional control

Bacterial flagella biosynthesis
FlhD4C2
s28
s70
s28
FlgM
FliA(s28)
FlhD4C2
FlgM
s
70
FlgM
hook
basal body
FlhDC activates a set of early promoters
1755

36. Examples of the transcriptional control

Bacterial flagella biosynthesis
FlgM
FlhD4C2
s
FlgM
28
hook
basal body
FlgM
FlhD4C2
s28
s28
FliA(s )
28
s
28
FliС(flagellin)
Positive feedback loop starts after hook/basal body assembly
1755

37. Examples of the transcriptional control

Bacterial flagella biosynthesis
FlhD4 C2
s
28
s28
FliA(s )
28
FliС( flagellin)
s28
FliС( flagellin)
Flagellin biosynthesis is activated after assembly of a functional
hook and basal body
1755

38. Examples of the transcriptional control

Transcriptional control of phage l
Genome
tL1
NuI A...J
att int xis
tR2 tt
CIII N rex CI cro CII OP Q SR
PL
Pi/CII
tR1
PR
Po
PR’
PRE /CII PaQ /CII
1755

39. Examples of the transcriptional control

Transcriptional control of phage l
Early genes
tL1
NuI A...J
att int xis
tR1
tR2 tt
CIII N rex CI cro CII OP Q SR
PL
Pi/CII
PR
Po
PR’
PRE /CII PaQ /CII
Making a choice
Hunger
cAMP
HflA
CII
Lysogeny
1755

40. Examples of the transcriptional control

Transcriptional control of phage l
CII activates genes required for lysogeny
tL1
NuI A...J
att int xis
tR2 tt
CIII N rex CI cro CII OP Q SR
PL
Pi/CII
tR1
PR
Po
PR’
PRE /CII PaQ /CII
1755

41. Examples of the transcriptional control

Transcriptional control of phage l
CI inactivate everything but its own gene
tL1
NuI A...J
att int xis
tR1
CIII N rex CI cro CII OP Q SR
PL PRM PR
Pi/CII
tR2 tt
Po
PR’
PRE /CII PaQ /CII
1755

42. Examples of the transcriptional control

Transcriptional control of phage l
CI protein: positive and negative feedback loops
PRM
RNAP
CI
ORIII ORII
PRM
CI
CI
ORIII ORII
PR
CI
ORI
PR
CI
ORI
1755

43. SOS response

Activated RecA cleaves LexA repressor
LexA
genes encoding
SOS response
components
LexA
RecA*
genes encoding
SOS response
components
LexA
And so
what?
1755

44. Examples of the transcriptional control

Transcriptional control of phage l
Activated RecA cleaves CI
RecA*
CI
PRM
PR
ORIII ORII
ORI
1755

45. Examples of the transcriptional control

Transcriptional control of phage l
Cro inactivate CI expression
(positive feedback loop = all-or-non decision)
PRM
cro
ORIII ORII
RNAP
PR
ORI
1755

46. Examples of the transcriptional control

Transcriptional control of phage l
Cro re-starts lytic pathway
tL1
NuI A...J
att int xis
tR1
CIII N rex CI cro CII OP Q SR
PL PRM PR
Pi/CII
tR2 tt
Po
PR’
PRE /CII PaQ /CII
1755

47. Examples of the transcriptional control

Transcriptional control of phage l
Expression of genes necessary for replication
tL1
NuI A...J
att int xis
tR1
tR2tt
CIII N rex CI cro CII OP Q SR
PL
Pi/CII
PR
Po
PR’
PRE /CII PaQ /CII
… and Q
1755

48. Examples of the transcriptional control

Transcriptional control of phage l
Q inactivate tt terminator
tL1
NuI A...J
att int xis
tR2 tt
CIII N rex CI cro CII OP Q SR
PL
Pi/CII
tR1
PR
Po
PR’
PRE /CII PaQ /CII
… and allows expression of genes necessary for virion packing and
cell lysis
1755

49. Examples of the transcriptional control

Transcriptional control of phage T4
ADP-ribosilation of a-CTD abolish interaction with UP elements
and protein activators
Alt
76kD
ADP
a
a
b’
s70
b
1755

50. Examples of the transcriptional control

Transcriptional control of phage T4
-Further ADP-ribosylation of a-CTD
- AsiA inhibit -35 box recognition
-MotA interacts with middle gene promoters
Mod
-Alc blocks transcription on DNA without 5hmC
AsiA
s70
MotA
mot
box
TATA
24kD
a
a
b’
b
ADP
ADP
Alc
19kD
1755

51. Examples of the transcriptional control

Transcriptional control of phage T4
Late genes use gp55/gp33 sigma and sliding clamp as enhancer
gp44/62
gp33
gp55
a
a
b’
b
ADP
ADP
Alc
3x gp45
1755

52. Examples of the transcriptional control

Transcriptional control of phage T4
T4 late transcription activation via sliding clamp
1755