Transcript No Slide Title

Regulation after initiation
Antitermination of transcription: l
Attenuation in biosynthetic
operons: trp
lac regulatory region
Activator binding site
Promoter
Operator
UV5 mutation, up TATAAT
-72
-52
TTTACA
TATGTT
-35
-10
a
cAMP-CAP
s
b
RNA polymerase
+1
b'
+11
Repressor
The CTD of the alpha subunit of RNA
Pol can interact with activators
aCTD
Class I promoters:
CAP binding sites
upstream of -35,
E.g. centered at -62,
-83, -93.
Class II promoters:
CAP binding sites
centered at -42,
Overlaps -35 box.
Binding of repressor blocks transcription
from pR but activates pRM
PR
-35
-10
oR3
N
2 dimers of
Repressor,
bound
cooperatively
cro
RNA Pol
-10
oR2
oR1
-35
= operator
PRM
-35
-10 = promoter
Antitermination occurs at two stages
in the l life cycle
Immediate early transcription
Transcription by E. coli RNA polymerase initiates at strong
promoters PR , PR’, and PL , and terminates at t’s.
att
int
gam
red
xis cIII N
Pint
tL1
cI
cro
cII O P Q
PL oL PRM PR tR1 PRE
oR
SR
tR2 PR‘ t6S
tR3
6S RNA
N
Cro
A…J
Antitermination by N protein leads to early
gene expression
N
att
int
gam
red
xis cIII N
Pint
tL1
N
cI
PL
cro
N
cII O P Q
PRM PR tR1 PRE
SR
A…J
tR2 PR‘ t6S
tR3
6S RNA
N protein
CIII
Recombination proteins
Cro
CII
Q protein
Replication proteins
Lytic cascade: Cro turns off cI, Q protein
action leads to late gene expression
Cro
att
int
gam
red
xis cIII N
Pint
tL1
Cro
cI
cro
Q
cII O P Q
PL oL PRM PR tR1 PRE
oR
SR
A…J
tR2 PR‘ t6S
tR3
Lytic functions
Replication proteins
Viral head & tail proteins
Review of r-dependent termination
of transcription
Termination of transcription in E. coli:
Rho-dependent site
5' ...AUCGCUACCUCAUAUCC GCACCUCCUCAAACGC UA CCUCGACCAGAAAGGCGUCUCUU
Termination occurs at one
of these 3 nucleotides.
• Little sequence specificity: rich in C, poor in G.
• Requires action of rho (r ) in vitro and in vivo.
• Many (most?) genes in E. coli have rho-dependent
terminators.
r he xam e r binds to prote in-fr e e
RNA and m ove s along it.
a
Model
for
action
of rho
factor
r
a
b
r-de pe nde nt s ite
b'
RNA polym e r as e tr ans cr ibe s along the
tem plate , andr m ove s along the RNA.
RNA polym e r as e paus e s at the
r-de pe nde nt ter m inator s ite ,
and r catche s up
Str uctur e in RNA that caus e s paus in
r u nw inds the RNA-DNA hybr id
and trans cription te rm inate s
Components needed for antitermination
• Sites on DNA
– nut sites (N utilization sites) for N protein, qut sites for
Q protein
– Are found within the transcription unit
– nut sites are 17 bp sequences with dyad symmetry
• Proteins
– Antiterminators: N protein and Q protein encoded by l
– Host proteins (encoded by E. coli)
• Nus A (encoded by nusA, N-utilization substance)
• Rho protein
Arrangement of nut sites in
transcription units
delayed
early txn (N
present)
immediate
early txn
PP RR
tL1
... cIII
nutL
N
PL
PL
cI
immediate
early txn
delayed
early txn (N
present)
cII
cro
nutR
tR1
O
P
Q
tR2
...
r-de pe nde nt s ite
N plus
Nus
factors
block
rho
action
r
N NusA
RNA polymerase (with N and NusA )
transcribes along the template, and r
mov es along the RNA.
RNA polym e r as e doe s NOT paus e at
the r-de pe nde nt te rm inator s ite , and
r ne ve r catche s up
Tr ans cr iption continue s pas t the
ter m inator
NusG and elongation
• NusG is another E. coli protein needed for lambda
N to prevent termination
• Homolog of a family of proteins involved in
elongation in prokaryotes and eukaryotes
• Eukaryotic DSIF
– DRB-sensitivity inducing factor (Flies and mammals)
• DRB is a drug that blocks transcriptional elongation
– Two subunits
• 160 kDa, homolog to yeast Spt5
• 14 kDa, homolog to yeast Spt4
– Implicated in positive and negative control of elongation
Regulation of E. coli trp operon by
attenuation of transcription
Organization of the E. coli trp operon
p,o
trpE
trpD
trpC
trpB
t t’
trpA
leader
attenuator
Chorismic
acid
tryptophan
COOH
CH2
CH2
O
OH
C
COOH
N
CH
NH2
COOH
The trp operon is regulated in part by an
apo-repressor
p,o
p o
trpE
trpD
trpE
trpC
trpB
trpA
t t’
Operon ON
p
o
trpE
+ trp
Apo-repressor
Repressor
(with trp bound)
Operon OFF
The trp operon is also regulated by attenuation
leader atten. trpE
p,o
1
RNA
27
AUG
trpD
trpC
54
70
UGGUGG
trp trp
Leader peptide:
14 amino acids,
2 are trp
trpA
t t’
114 126
140
trpB
90
UGA
txn
pause
attenuator
Rho-independent terminator
of transcription.
Conditional :
Terminates in high [trp],
Allows readthrough in
low [trp]
Termination of transcription in E. coli:
Rho-independent site
AG U U
U
A
G
G
A
A
UG
GC
GC
C GA
C U
UA
UA
GC
G
A
CG
AU
AU
AU
GC
CG
CG
CG
UA
AU
A U U U U U ...3'
5' ... G C
G+C rich region in stem
Run of U's 3' to stem-loop
How attenuation works in trp
• The [trp] determines the [trp-tRNA].
• The [trp-tRNA] determines whether a translating
ribosome will add trp to the leader peptide.
• If trp is added:
– The ribosome moves on to the translation stop codon.
– This places the attenuator in a secondary structure that
causes termination of transcription (OFF).
• If trp is not added:
– A different secondary structure forms in the leader RNA
– Allows readthrough transcription into the structural
genes (ON).
Basic components for attenuation in trp
translation
[trp-tRNA] of trpL
High
complete
Low
stalls at
trp codons
secondary structures
formed in RNA
Attenuator Operon
3-4 stem
terminate txn OFF
2-3 stem
allow readON
through txn
Requirements for attenuation in trp
operon
• Simultaneous transcription and translation.
• A segment of RNA that can serve as a
terminator because of its base-paired
(secondary) structure.
• An alternative secondary structure in the
RNA that does not allow termination of
transcription.
• Does NOT need an additional protein, such
as a repressor.
Alternative base-paired structures in leader RNA
1
27
54
AUG
70
UGGUGG
trp trp
UGA
1
1
114 126
txn
pause
2
140
attenuator
3 4
2
3
Termination
of transcription
90
4
1
No termination
2
3
4
Progress of ribosome determines
secondary structure of trp leader RNA
attenuator
trp trp
ribosome
UGGUGG UGA
1
UGGUGG 2
1
3
High [trp],
4 termination
of transcription
2
3
4
Low [trp],
No termination
Examples of mutational analysis of trp
• Translation of trp leader is needed for regulation
– Mutation of AUG prevents transcription past the
attenuator
– Without translation, the 1:2 and 3:4 stem-loops form,
and thus causing termination
• Specific secondary structures are needed
– Mutations that decrease the number of base pairs in the
3:4 stem-loop increase expression (less termination) in
high [trp].
– Compensatory mutations that restore the wild-type
number of base pairs allow termination in high [trp].
Many biosynthetic operons are
regulated by attenuation
• Amino acid biosynthetic operons
• E.g., his, phe, leu, thr, ilv
• In each case, a short leader RNA and
polypeptide precede the structural genes.
This leader polypeptide is rich in the amino
acid that is the product of the pathway.