molecular biology and biochemistry 694:408 / 115:512 spring 2007, lectures 13-14
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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) - PowerPoint PPT PresentationTRANSCRIPT
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
A/T rich
Up element
αCTD
α NTDβ
σ
β'
-35 -10UP
Transcription from some promoters is initiated by alternative sigma () factors
β
70
β'
-35 -10
α β
σ32
β'
-35 -10
α
Heat Shock GenesMost Genes
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 CCGATAT
s37 used in sporulation AGGNTTT GGNATTGNT
s32 used in sporulation AAATC TANTGTTNTA
s29 synthesized in sporulation TTNAAA CATATT
gp28SPO1 middle expression AGGAGA TTTNTTT
gp33-34 SPO1 late expression CGTTAGA GATATT
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 CCGATAT
s37 used in sporulation AGGNTTT GGNATTGNT
s32 used in sporulation AAATC TANTGTTNTA
s29 synthesized in sporulation TTNAAA CATATT
gp28SPO1 middle expression AGGAGA TTTNTTT
gp33-34 SPO1 late expression CGTTAGA GATATT
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Bacteriophage - "eaters of bacteria"
Phage Early gene 28
Phage Mid. genes 33 34
Early
Middle
Transcription of phage SPO1 genes
RNAP70
RNAP28
RNAP28
Phage Late genes
LateRNAP
33 34
RNAP70 28
33 34RNAP33 34
RNAP28
Genetic regulation
lac system of E. coli
“What’s true for E. coli is true for an elephant.”
J. Monod
β-Gal is produced only when lactose is present
β-gal induction can be due to
1. Activation of preexisting enzyme (i.e., removal of repressor)
2. Synthesis of new enzyme
Gratuitous inducers do not act as substrates
Lactose is both an inducer and a substrate for β-Gal
Some substrates do not work as inducers
Action of the enzyme on the inducer is neither necessary nor sufficient for induction
Induction kinetics of β-Gal under gratuitous conditions
p = (amount of β-Gal)/(total cell protein)
lac system: transcription regulation
Regulation of Transcription
1. Transcription initiation/RNA synthesis
2. mRNA Turnover RNAP
1
2mRNA
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
21
The lac locus of E. coli
β-Galgalactos
ide permease
galactoside
transacetylase
lacY mutants are cryptic
lacI mutants are constitutive (first example of mutants that affect production, not activity)
lacA mutants are Lac+
lacZ mutants are Lac-
The PaJaMo experiment
Hfr lacI+ lacZ+ StrS TsXS x F- lacI- lacZ- StrR TsxR
Set a cross in the absence of inducer:
After some time, kill the donor with Str and T6
Monitor β-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
DNA
lacIRepressor
Inducer
The presence of inducer changes the conformation of LacI repressor so that it can no longer bind DNA
Distinction between factors (proteins) and elements (DNA sites)
R e g u l a t o r
XR e g u l a t o r
R e g u l a t o r
X
R e g u l a t o rR e g u l a t o r
XR e g u l a t o r
X
ii) Regulatory elements act in cis
i) Regulatory factors act in trans
The LAC OPERON
LacI binds DNA as a tetramer to better repress transcription
Why did Jacob & Monod not find O2 and O3?
X-gal
White
Blue
White
Blue
White
Genetic analysis of the LacI binding sites
O3 O1 O2
P lacZRepression
1300
1.0
1.0
1.0
O3 O1 O2
O3 O1 O2
O3 O1 O2
440O3 O1 O2
700O3 O1 O2
1.9O3 O1 O2
18O3 O1 O2
Glucose effect: no response to inducers in the presence of glucose
glucose energy
glycerolpgi
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)
Catabolism
???
Catabolite repression occurs for a wide range of sugars
Catabolite repression mutants must therefore be defectivein 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
cAMP level highwhen glucose is low
codes for CAP (catabolite activating protein).
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
CAP RNAP LacI
RNAP
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
Lac
AroH
Gal
Repressor Operator Sites CAP Operator Sites
Lac
Trp
AroH
TrpR
Gal
β
β'
-35 -10
D
Act.
λPRM- cI
αCTD αNTD
α CTDαNTD β
σ
β'
-35 -10
CAct.
lac-CAP
αCTD
α NTDβ
σ
β'
-35 -10
B
UPrrnB
β
σ
β'
-35 -10
AαNTDαCTD
lacUV5
E
α CTD αNTD β
σ
β'
-35 -10
Act.Act.
CAP & cI
Different mechanisms of transcriptional activation
A) Strong promoters
B) Promoters with UP elements
C) Activation through interactions with the αCTD
D) Activation through interactions with other components of RNAP
E) Activation through interactions with components multiple components of RNAP by multiple activators
Different types of negative and positive control of transcription
Changes in DNA topology affect isomerization step in formation of the open complex
RNAP
DNA
OpenComplex
ClosedComplex
KB ki
-35 -1015-17 bp
RNAP
-35 -1019 bp
merT
AverageProm.
Mechanism of activation by MerR
MerR
merT-35 -10
Hg++
MerR
17 bp
RNAP
Enzyme repression: the trp operator
The synthesis of Trp structural genes is controlled by unlinkedTrpR 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
precursor Trp
TrpE TrpD TrpB
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
The existence of c and vir mutants. are immune to c, but not vir
Immunity of lysogens to superinfection with wt
Zygotic induction