scotty merrell department of microbiology and immunology b4140 [email protected]
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Scotty Merrell Department of Microbiology and Immunology B4140 [email protected] Regulation of Gene Expression I. QUESTIONS. 1.Why does the expression of genes need to be regulated?. 2.Why is it important to study gene regulation?. 3.How is the expression of genes regulated?. - PowerPoint PPT PresentationTRANSCRIPT
Scotty MerrellDepartment of Microbiology and Immunology
Regulation of Gene Expression I
1. Why does the expression of genes need to be regulated?
QUESTIONS
3. How is the expression of genes regulated?
2. Why is it important to study gene regulation?
4. How do we study gene regulation?
Pathogenic bacteria:External reservoir Host
Infection site #1 Infection site #2
Bacteria experience different conditions depending on environment
1. Why does the expression of genes need to be regulated?
QUESTIONS
3. How is the expression of genes regulated?
2. Why is it important to study gene regulation?
4. How do we study gene regulation?
Pathogenic bacteria produce virulence factors whenthey sense they are inside of a host
Vibrio cholerae, the cause of cholera, produces toxin insideof the host. Understanding regulation of expression of this toxin
is a means of understanding ways to prevent its production.
ICDDR,B
1. Why does the expression of genes need to be regulated?
QUESTIONS
3. How is the expression of genes regulated?
2. Why is it important to study gene regulation?
4. How do we study gene regulation?
DNAPromoteroperator
Attenuator Stop signal
RNA polymeraseRegulatory proteins aa-tRNAs RNA polymerase
Transcription
Transcriptional control(a) Transcription initiation: positive/negative(b) Transcription termination: attenuation/anti-termination
Regulation of gene expression
mRNA
Degradation
Translational controlTranslation initiation: positive/negative
Ribosomebindingsite
Stop signal
Regulatory proteinsAntisense RNAs
Translation
Protein
Post-translational control(e.g., proteolysis)
Regulation of Gene Expression
DNAPromoteroperator
Attenuator Stop signal
RNA polymeraseRegulatory proteins aa-tRNAs RNA polymerase
Transcription
Transcriptional control(a) Transcription initiation: positive/negative(b) Transcription termination: attenuation/anti-termination
Regulation of gene expression
mRNA
Degradation
Translational controlTranslation initiation: positive/negative
Ribosomebindingsite
Stop signal
Regulatory proteinsAntisense RNAs
Translation
Protein
Post-translational control(e.g., proteolysis)
Regulation of Gene Expression
’
5’ppp
Promoter
Polymerase binds topromoter region, forminga closed complex
Polymerase unwindsDNA, forming anopen complex
Transcription begins
Core enzyme
Holoenzyme
TTGACAAACTGT
TATAATATATTA
-6-13-30-37 +1-10 region-35 region
mRNA
’
’
Transcription initiation
RNA polymerase-promoter interactions
Some promoters contain UP elements that stimulate transcriptionthrough direct interaction with the C-terminal domains of the
subunits of the RNA polymerase
Promoter with a full UP element containing two consensus subsites.
Promoter with an UP element containing only a consensus proximal subsite.
Promoter with an UP element containing only a consensus distal subsite.
Arrangement of subunits on UP elements
Genes come in two main flavors:
1. Constitutively expressed (transcription initiationis not regulated by accessory proteins)
2. Regulated (transcription initiationis regulated by accessory proteins)
a. Negatively Regulated--Repressor Proteinb. Positively Regulated--Activator Protein
Mechanisms of Regulation of Transcription Initiation:Negative Regulation
RNA Polymerase
Mechanisms of Regulation of Transcription Initiation:Negative Regulation
Repressor
Repressor
Co-repressor
Repressor
Inactivator
The lac operona model for negative regulation
A bacterium's prime source of food is glucose, since it does not have to be modified to enter the respiratory pathway. So
if both glucose and lactose are around, the bacterium wants to turn off lactose metabolism in favor of glucose metabolism.
There are sites upstream of the lac genes that respond to glucose concentration.
This assortment of genes and their regulatory regions is called the lac operon.
H O
L a c t o s e
O
H O C H 2O
O H
O H
H O C H 2O
O H
O H
O H
H O C H 2O
O H
O H
O H
G a l a c t o s e
H O C H 2O
H OO H
O H
O H
G l u c o s e
+
- G a l a c t o s i d a s e
- G a l a c t o s i d a s e
OH OH O C H 2
O
O H
O H
C H 2O
O H
O H
O H
H O
A l l o l a c t o s e
Structural genes:lacZ encodes -galactosidase lacY encodes -galactoside permeaselacA encodes -galactoside transacetylase
Regulatory gene and elements:lacI --- encodes repressor proteinlacO --- operatorlacP --- promoter
lacIPi P O lacYlacZ lacA
The lac operon
The lac promoter and operator regions
Lac Repressor(monomer) (tetramer)
The Lac Repressor is constitutively expressed
Repressor binding prevents transcription
When lactose is present, it acts as an inducer of the operon. It enters the cell and binds to the Lac repressor, inducing a conformational
change that allows the repressor to fall off the DNA. Now the RNA polymerase is free to move along the DNA and RNA can be made from
the three genes. Lactose can now be metabolized.
Remember, the repressor actsas a tetramer
When the inducer (lactose) is removed, the repressor returns to its original conformation and binds to the DNA, so that RNA polymerase
can no longer get past the promoter to begin transcription. No RNA and no protein are made.
Remember, the repressor actsas a tetramer
1. Mutation in the regulatory circuit may either abolish expression of the operon or cause it to occur without responding to regulation.
2. Two classes of mutants: A. Uninducible mutants: mutants cannot be expressed at all. B. Constitutive mutants: mutants continuously express genes that do not respond to regulation.
3. Operator (lacO): cis-acting element Repressor (lacI): trans-acting element
How to identify the regulatory elements?
cis-configuration: description of two sites on the same DNA molecule (chromosome) or adjacent sites.
cis dominance: the ability of a gene to affect genes next to it on the same DNA molecule (chromosome), regardless of the natureof the trans copy. Such mutations exert their effect, not because of altered products they encode, but because of a physical blockage or inhibition of RNA transcription.
trans-configuration:description of two sites on different DNA molecules (chromosomes) or non-contiguous sites.
Definitions:
Constitutive mutants: do not respond to regulation.
l a c I -P i P O l a c Yla c Z l a c A
N o n b in d i n gr e p r e s s o r
m R N A m R N AX
M u t a t i o n s t h a t i n a c t i v a t e t h e l a c I g e n e ( l a c I - ) c a u s e t h e o p e r o n t o b e c o n s t i t u t i v e l y e x p r e s s e d , b e c a u s e t h e m u t a n t r e p r e s s o rp r o t e i n c a n n o t b i n d t o t h e o p e r a t o r .
Would this be a cis-dominant or recessive mutation?
lacI+Pi
mRNA
lacI-Pi P O lacYlacZ lacA
mRNA
Constitutive mutants in the lacIgene are recessiveConstitutive mutants can be recessive
lacI+
mRNA
lacI-Pi P O lacYlacZ lacA
mRNA mRNAX
Constitutive mutants can also be dominant if the mutant allele produces a “bad” subunit, which is not only itself unable to bind to
operator DNA, but is also able to act as part of a tetramer to prevent any “good” (wild type LacI) subunits from binding.
et al.
Think about how you could determinewhether a mutation was dominant or
recessive.
Questions about negativeRegulation of lac ?
Mechanisms of Regulation of Transcription Initiation:Positive Regulation
RNA Polymerase
Mechanisms of Regulation of Transcription Initiation:Positive Regulation
RNA Polymerase
Activator
The lac operona model for positive regulation
When levels of glucose (a catabolite) in the cell are high, a molecule called cyclic AMP is inhibited from forming. So
when glucose levels drop, more cAMP forms. cAMP binds to a protein called CAP (catabolite activator protein), which is
then activated to bind to the CAP binding site. This activates transcription, perhaps by increasing the affinity of the site for
RNA polymerase. This phenomenon is called catabolite repression, a misnomer since it involves activation, but
understandable since when it was named, it seemed that the presence of glucose repressed all the other sugar
metabolism operons.
CAP --- a positive regulator
1. Catabolite repression: the decreased expression of many bacterial operons that results from addition of glucose. Also known as “glucose effect” or “glucose repression”.
2. E. coli catabolite gene activator protein (CAP; also known as CRP, the cAMP receptor protein).
3. CAP-cAMP activates more than 100 different promoters, including promoters required for utilization of alternative carbohydrate carbon sources such as lactose, galactose, arabinose, and maltose.
Inactive CAP
CAP --- a positive regulator
A. under catabolite-respressing conditions cAMP level is very low
crp Target operon
cAMP
B. Under non-catabolite-respressing conditions cAMP level is very high
InactiveCAP
CAP-cAMPActive CAP
crp Target operon
RNAP RNAP
cAMPAut
oreg
ulat
ion
Act
ivat
ion
-O-P~O-P~O-P-O-CH2
=-
O
O
=-
O
O
=-
O
OO
C
--
H C
--
OHC
--
OH
C
--
HH H
Adenine
ATP
O-CH2 OC
--
H C
--
OC
--
OH
C
--
HH H
O=PO-
Adenine
cAMP
Adenylate cyclase
PTSGlucose Glucose-6-P
IIAGlc-P
IIAGlc
OUT IN
How does glucose reduce cAMP level?
1. IIAGlc-P activates adenylate cyclase.2. Glucose decreases IIAGlc-P level, thus reducing cAMP production.3. Glucose also reduces CAP level: crp gene is auto-regulated by CAP-cAMP.
PTS - phosphoenolpyruvate-dependent carbohydrate phosphotransferase systemIIAGlc - glucose-specific IIA protein, one of the enzymes involved in glucose transport.
Activation of expression of the lac operon
E. coli CAP (CRP) --- 209 amino acids
NH2- -COOH
140-209
DNA-bindingHelix-turn-helix
AR1156-164
His19His21
Glu96Lys101
AR2
Dimerization and cAMP-binding
1-139
Transcription activation by CAP at class I CAP-dependent promoters
(-62)
Transcription activation:1. Interaction between the AR1 of the downstream CAP subunit and one copy of CTD.2. The AR1-CTD interaction facilitates the binding of CTD to the DNA downstream of CAP.3. Possibly, interaction between same copy of CTD and the bound at the –35 element.4. The interaction between the second CTD and CAP is unclear.
The result: increasing the affinity of RNAP for promoter DNA, resulting in an increase in the binding constant KB, for the formation of the RNAP-promoter closed complex
Transcription activation by CAP at class I CAP-dependent promoters (cont.)
(-103, -93, -83, or –72)
Transcription activation: Possibly, the second copy of CTD may interact with the DNA downstream of CAP, and may interact with the bound at the –35 element.
Results: increasing the affinity of RNAP for promoter DNA, resulting in an increase in the binding constant KB, for the formation of the RNAP-promoter closed complex
Transcription activation by CAP at class II CAP-dependent promoters (cont.)
(-42)
Transcription activation:1. Interaction between the AR1 of the upstream CAP subunit and one copy of CTD
(either CTDI or CTDII, but preferentially CTDI). The AR1-CTD interaction facilitates the binding of CTD to the DNA upstream of CAP.
Results: increase in the binding constant KB, for the formation of the RNAP-promoter closed complex
• Interaction between the AR2 of the downstream CAP subunit and NTDI.
Result: increase the rate constant, kf, for isomerization of closed complex to open complex.
Transcription activation by CAP at class III CAP-dependent promoters
(-103 or –93) (-62)
Transcription activation:
Each CAP dimer functions through a class I mechanism with AR1 of the downstream subunit of each CAP dimer interacting with one copy of CTD
Results: synergistic transcription activation
Transcription activation by CAP at class III CAP-dependent promoters (cont.)
(-103, -93, or -83) (-42)
Transcription activation:
The upstream CAP dimer functions by a class I mechanism, with AR1 of the downstream subunit interacting with one copy of CTD; the downstream CAP dimer functions by a class II mechanism, with AR1 and AR2 interacting with the other copy of CTD and NTD, respectively.
Results: synergistic transcription activation
(a) Glucose present (cAMP low); no lactose;
lacIPi P O lacYlacZ lacA
Repressormonomer
Repressortetramer
mRNAX
(b) Glucose present (cAMP low); lactose present
Repressormonomer
Repressortetramer
mRNA
Inducer
High level of mRNAX
Inactiverepressor
High
(c) No glucose (cAMP high); lactose present
cAMPCAP
Glucose effect on the E. coli lac operon
lacIPi P O lacYlacZ lacA
Repressormonomer
Repressortetramer
mRNAX
lacIPi P O lacYlacZ lacA
Repressormonomer
Repressortetramer
mRNAX
No lactose inside the cells!(inducer exclusion)!
(a) Glucose present (cAMP low); no lactose;
lacIPi P O lacYlacZ lacA
Repressormonomer
Repressortetramer
mRNAX
(b) Glucose present (cAMP low); lactose present
Repressormonomer
Repressortetramer
mRNA
Inducer
High level of mRNAX
Inactiverepressor
High
(c) No glucose (cAMP high); lactose present
cAMPCAP
Glucose effect on the E. coli lac operon
lacIPi P O lacYlacZ lacA
Repressormonomer
Repressortetramer
mRNAX
lacIPi P O lacYlacZ lacA
Repressormonomer
Repressortetramer
mRNAX
No lactose inside the cells!(inducer exclusion)!
Inducer exclusion: How does it work?
1. Uptake of glucose dephosphorylates enzyme IIglc.
2. Dephosphorylated enzyme IIglc binds to and inhibits lactose permease.
3. Inhibition of lactose permease prevents lactose from entering the cell.
4. Hence, the term inducer exclusion.
Questions about positive regulationof the lac operon?
Dual positive and negative control of transcription initiation:
the E. coli ara operon
The E. coli L-arabinose operon
+
+
AraC exists in two states
P1 P2
Arabinose
Arabinose
ActivatorAntiactivator
AraC acts as a positive or negative regulator based on its conformational state and binding affinity for
various sites in the two promoter regions.
AraC encodes the regulatorAraO1 and AraO2 encode operatorsCAP is a CAP binding siteAraI is an additional regulatory regionAraBAD are the structural genes
If AraC concentration becomes too high, AraC will also bind to AraO1 and repress its own expression.
No arabinose
+ arabinose
In the absence of arabinose, the P1 form of AraC binds AraO2 and AraI to prevent any P2 form from binding and activating expression--this is anti-activation, not repression!
In the presence of arabinsose, AraC shifts to the P2 form and bindsAraI and acts to activate transcription.
Therefore AraC is an Activator, Repressor and Anti-activator!!
The regulatory regions of the PC and PBAD promoters
The domain structure of one subunit of the dimeric AraC protein
The PC and PBAD Regions in the presence or absence of arabinose
+ L-arabinose
Hypothetical model of the activation of the PBAD promoter
1. PBAD – class II promoter2. Possible interactions: between the CTD of RNAP
and the CAP protein and AraC protein and DNA
1. Find mutations that render the regulation uninducible or constitutive.
2. Decide by performing a complementation test if the mutants are dominant or recessive.
3. If they are recessive, decide if the system is regulated by repression or by activation. A recessive mutated activator has most likely lost function: the system will become uninducible. A recessive mutated repressor has also lost function, but now the system will show constitutive expression.
4. Decide if the elements of the system act in cis or in trans to each other: are they proteins or DNA binding sites?
5. Construct a model.
Strategies for Understanding Regulation
Questions about ara regulation?
A. Transcriptional control1. Transcription initiation a) Positive b) Negative2. Transcription termination Attenuation
B. Translational control1. Positive2. Negative
C. Post-translational control--Proteolysis
Regulatory mechanisms used to control gene expression
DNAPromoteroperator
Attenuator Stop signal
RNA polymeraseRegulatory proteins aa-tRNAs RNA polymerase
Transcription
Transcriptional control(a) Transcription initiation: positive/negative(b) Transcription termination: attenuation/anti-termination
Regulation of gene expression
mRNA
Degradation
Translational controlTranslation initiation: positive/negative
Ribosomebindingsite
Stop signal
Regulatory proteinsAntisense RNAs
Translation
Protein
Post-translational control(e.g., proteolysis)
RNAP
Transcription termination players:Termination sequence
RNA polymeraseand sometimes the Rho ( factor
A B C D
Promoter Operon of 4 genes Terminator
X
Rho-independentterminator
Rho-independentterminator
Rho-dependentterminator
Two major types of Terminator Sequences1. Rho-independent2. Rho-dependent
Attenuation: Premature termination of transcription of operons for amino acid biosynthesis
(trp, his, leu, etc.)
Relies on coupled transcription and translation and RNA secondary structure
P/O trpDtrpE trpC
mRNA
Tryptophanrepressor
mRNA
L trpAtrpBP/O trpR
1
23
4
The trp leader mRNA encodes the LEADER PEPTIDE
MetLysAlaIlePheValLeuLysGlyTrpTrpArgThrSer5’-AUGAAAGCAAUUUUCGUACUGAAAGGUUGGUGGCGCACUUCC U CCCAUAGACUAACGAAAUGCGUACCACUUAUGUGACGGGCAAAGA GCCCGCCUAAUGAGCGGGCUUUUUUUUGAACAAAAUUAGAGA-3’
Organization of Tryptophane Biosynthesis Genes
End product of the pathway
1 2 3 4
mRNA forms secondary structures
Adapted from http://www.andrew.cmu.edu/user/berget/Education/attenuation/atten.html
3 and 4 form a Rho-independent
terminator
Two possible alternative structures can form2 is complementary to 1 and 33 is complementary to 2 and 4
2 and 3 form the Pre-emptor, which prevents
Terminator formation
Pre-emptor
Tryptophan absent Tryptophan present
UGGUGGCGCACUUCCU
UGGUGGCGCACUUCCU
RegulatoryOperon Leader Peptide Sequence Amino Acid(s) his Met-Thr-Arg-Val-Gln-Phe-Lys-His-His-His-His -His-His-His-Pro-Asp His pheA Met-Lys-His-Ile-Pro-Phe-Phe-Phe-Ala-Phe-Phe -Phe-Thr-Phe-Pro Phe leu Met-Ser-His-Ile-Val-Arg-Phe-Thr-Gly-Leu-Leu -Leu-Leu-Asn-Ala-Phe-Ile-Val-Agr-Gly-Agr-Pro -Val-Gly-Gly-Ile-Gln-His Leu thr Met-Lys-Agr-Ile-Ser-Thr-Thr-Ile-Thr-Thr-Thr -Ile-Thr-Ile-Thr-Thr-Gly-Asn-Gly-Ala-Gly Thr, Ile
ilv Met-Thr-Ala-Leu-Leu-Arg-Val-Ile-Ser-Leu-Val -Val-Ile-Ser-Val-Val-Val-Ile-Ile-Ile-Pro-Pro -Cys-Gly-Ala-Ala-Leu-Gly-Arg-Gly-Lys-Ala Leu, Val, Ile
Biosynthetic Operons Regulated by Attenuation
Attenuation can also occur at the level of Protein-RNA interaction:
Regulation of the trp operon in Bacillus
Model of trp transcriptional
control
Binding of activated TRAP
in the leaderpeptide
results in the formation of a
terminatorstructure
Take home message:
Transcription of genes to produce mRNAcan be controlled at the level of
initiation and/or termination
STOP