Molecular Biology and Biochemistry
694:408 / 115:512
Spring 2007, Lectures 13-14
Regulation of prokaryotic transcription
Watson et al., (2004) Mol. Biol. Of the Gene, Chapter 16
Garrett and Grisham, Biochemistry (2005), Chapter 29 (pg. 942-974)
Lodish et al., (2000) Mol. Cell Biol. Chapter 10 (pg. 342); Chapter 12 (pg. 485-491)
Lewin (2000), Genes VII, Chapter 9; Chapter 10
Strong promoters contain close matches to the consensus site
Up element
A/T rich
 NTD
CTD
UP


-35
-10
'
Transcription from some promoters is initiated by
alternative sigma () factors
Most Genes


Heat Shock Genes
'


-35
-10


-35
-10
'
Different  factors in Bacillus subtilis are used at
different stages of growth (vegetative vs. sporulation)
Sigma
Source & Use
-35 region -10 region
s43
vegetative: general genes
TTGACA TATAAT
s28
vegetative: flagellar genes
CTAAA
s37
used in sporulation
AGGNTTT GGNATTGNT
s32
used in sporulation
AAATC
TANTGTTNTA
s29
synthesized in sporulation
TTNAAA
CATATT
gp28
SPO1 middle expression
AGGAGA TTTNTTT
gp33-34
SPO1 late expression
CGTTAGA GATATT
CCGATAT
Different  factors in Bacillus subtilis are used at
different stages of growth (vegetative vs. sporulation)
Sigma
Source & Use
-35 region -10 region
s43
vegetative: general genes
TTGACA TATAAT
s28
vegetative: flagellar genes
CTAAA
s37
used in sporulation
AGGNTTT GGNATTGNT
s32
used in sporulation
AAATC
TANTGTTNTA
s29
synthesized in sporulation
TTNAAA
CATATT
gp28
SPO1 middle expression
AGGAGA TTTNTTT
gp33-34
SPO1 late expression
CGTTAGA GATATT
CCGATAT
Bacteriophage - "eaters of bacteria"
QuickTi me™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Transcription of phage SPO1 genes
Early
Middle
70
RNAP
Phage Early gene 28
28
70
RNAP
28
28
RNAP
Phage Mid. genes 33 34
34
28
33
Late
33
34
RNAP
RNAP
33
34
Phage Late genes
Genetic regulation
lac system of E. coli
“What’s true for E. coli is true for an elephant.”
J. Monod
-Gal is produced only when lactose is present
-gal induction can be due to
1. Activation of preexisting enzyme (i.e., removal of repressor)
2. Synthesis of new enzyme
Lactose is both an inducer and a substrate for -Gal
Gratuitous inducers do not act as substrates
Some substrates do not work as inducers
Action of the enzyme on the inducer is neither necessary
nor sufficient for induction
Induction kinetics of -Gal under gratuitous conditions
p = (amount of -Gal)/(total cell protein)
lac system: transcription regulation
Regulation of Transcription
1. Transcription initiation/RNA
synthesis
2. mRNA Turnover
RNAP
1
mRNA
2
Selection of Lac- mutants (negative selection nutritional marker)
+Lac
Tricks
use chromogenic substrates (X-gal) and gratuitous inducers
(IPTG) to select for Lac mutants (Lac+ - blue, Lac- - white)
use diagnostic plates (EMB) to elect for absence of sugar
fermentation
1
2
The lac locus of E. coli
-Gal
lacZ mutants are LaclacY mutants are cryptic
galactoside
permease
galactoside
transacetylase
lacA mutants are Lac+
lacI mutants are constitutive (first example of mutants that
affect production, not activity)
The PaJaMo experiment
Set a cross in the absence of inducer:
Hfr lacI+ lacZ+ StrS TsXS x F- lacI- lacZ- StrR TsxR
After some time, kill the donor with Str and T6
Monitor -Gal in the presence or in the absence of inducer
The properties of lacO mutants provide genetic proof of operon model
lac operator
Most bacterial operator sequences are short inverted repeats;
Most transcription regulators are dimeric
The presence of inducer
changes the conformation of
LacI repressor so that it can
no longer bind DNA
DNA
lacI
Repressor
Inducer
Distinction between factors (proteins) and elements
(DNA sites)
i) Regulatory factors act in trans
Regulator
X
X
Regulator
Regulator
ii) Regulatory elements act in cis
Regulator
Regulator
X
Regulator
X
The LAC OPERON
LacI binds DNA as a tetramer to better repress transcription
Why did Jacob & Monod not find O2 and O3?
Genetic analysis of the LacI binding sites
P
lacZ
Repression
X-gal
O2
1300
White
O3
O1
O3
O1
O2
1.9
Blue
O3
O1
O2
440
White
O3
O1
O2
700
White
O3
O1
O2
18
Blue
O3
O1
O2
1.0
O3
O1
O2
1.0
O3
O1
O2
1.0
Glucose effect:
no response to inducers in the presence of glucose
Catabolism
???
glucose
energy
pgi
glycerol
pgi- mutants grown on glycerol induce lac genes
even in the presence of glucose
Interpretation: glucose effect is due a product of glucose catabolism
(catabolic repression)
Catabolite repression occurs for a wide range of sugars
Catabolite repression mutants must therefore be defective
in utilization of wide range of sugars (cells will be permanently
repressed).
Select on EMB agar.
Mutants defective in catabolite regulation occur in two distinct loci
cya
crp
codes for CAP
(catabolite activating
protein).
cAMP level high
when glucose is low
CAP, when bound to
cAMP, binds to lac
regulatory region and
activates transcription
of structural genes
LAC Operon and catabolite repression
Positive control of the lac operon is exerted by
cAMP-CAP Catabolite Activator Protein
Cooperative binding of cAMP-CAP and RNA polymerase to
the lac control region activates transcription
The lac control region contains three critical cis-acting sites
RNAP
CAP
RNAP LacI
lac operator: the regulatory region
CAP binding bends the DNA
Residues that interact
with RNAP
Operator sites can be in different places
with respect to the start of the promoter
Repressor Operator Sites
Lac
CAP Operator Sites
Lac
Trp
AroH
AroH
TrpR
Gal
Gal
A
Different mechanisms of
transcriptional activation
A) Strong promoters
CTD
NTD


lacUV5
-35
B
-10
 NTD
CTD
rrnB
B) Promoters with UP elements

-35
UP
Act.
 CTD
lac-CAP
-10

NTD
-35
E) Activation through interactions
with components multiple
components of RNAP by multiple
activators
 CTD Act. NTD
PRM- cI
-10

 CTD
-35
CAP & cI
NTD
Act. 
-35
'

-10
E
Act.
'

D
D) Activation through interactions
with other components of RNAP
'

C
C) Activation through interactions
with the CTD
'


-10
'
Different types of negative and positive control of
transcription
Changes in DNA topology affect isomerization
step in formation of the open complex
Closed
Complex
RNAP
Open
Complex
DNA
KB
ki
Mechanism of activation by MerR
RNAP
Average
Prom.
-35
-10
15-17 bp
merT
-35
MerR
19 bp
-10
Hg++
RNAP
merT
-35
MerR
17 bp
-10
Enzyme repression: the trp operator
The synthesis of Trp structural genes is controlled by unlinked
TrpR repressor. TrpR binds to Trp operator in the presence of Trp
(product inhibition).
Both trpR and trpO mutants are derepressed
Crossfeeding analysis of Trp mutants allows to analyze
the biochemistry of Trp biosynthesis pathway
TrpE
precursor
TrpD
TrpB
Trp
Attenuation of trp operator expression
attenuator
Deletions in the attenuator increase basal synthesis of Trp enzymes
the trp attenuator region
Attenuation occurs due to formation of alternative secondary
RNA structures in the leader sequence in the presence or absence or Trp
The  repressor idea
Zygotic induction
Immunity of lysogens to superinfection with wt
The existence of c and vir mutants.  are immune to c, but not vir