©2000 timothy g. standish ecclesiastes 3:1, 17 1to every thing there is a season, and a time to...
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©2000 Timothy G. Standish
Ecclesiastes 3:1, 171 To every thing there is a season, and a
time to every purpose under the heaven:17 I said in mine heart, God shall judge the
righteous and the wicked: for there is a time there for every purpose and for every work.
©2000 Timothy G. Standish
Controlling Controlling Gene Expression:Gene Expression:
BacteriaBacteriaTimothy G. Standish, Ph. D.
©2000 Timothy G. Standish
All Genes Can’t be Expressed All Genes Can’t be Expressed At The Same TimeAt The Same Time
Some genes are needed for the function of all cells all the time. These continually expressed genes are called constitutive genes.
Other genes are only needed by certain cells or at specific times. The expression of these inducible genes is tightly controlled.
For example, pancreas beta cells make the protein insulin by expressing the insulin gene. Neurons, in comparison, don’t express insulin.
©2000 Timothy G. Standish
Operons Are Groups Of Genes Operons Are Groups Of Genes Expressed By ProkaryotesExpressed By Prokaryotes
The genes grouped in an operon are all needed to complete a given task
Each operon is controlled by a single control sequence in the DNA
Because the genes are grouped together, they can be transcribed together then translated together
©2000 Timothy G. Standish
The The LacLac Operon Operon Genes in the lac operon allow E. coli bacteria to metabolize
lactose E. coli is unlikely to encounter lactose, so it would be
wasteful to produce the proteins needed to metabolize it unless necessary
Metabolizing lactose for energy only makes sense when two criteria are met:– Other more readily metabolized sugar (glucose) is unavailable
– Lactose is available
©2000 Timothy G. Standish
The The LacLac Operon - Parts Operon - Parts The lac operon is made up of a control region and four genes:
1 LacZ - -galactosidase - Enzyme that hydrolyzes the bond between galactose and glucose
2 LacY - Codes for a permease that lets lactose across the cell membrane
3 LacA - Transacetylase - An enzyme whose function in lactose metabolism is uncertain
4 Repressor - A protein that works with the control region to control expression of the operon
©2000 Timothy G. Standish
The The LacLac Operon - Control Operon - Control The control region is made up of two parts:
1 Promoter – Promoters are specific DNA sequences to which RNA Polymerase
binds so that transcription can occur– The lac operon promoter also has a binding site for a protein called
Catabolite Activator Protein (CAP)
2 Operator – The binding site of the repressor protein– The operator is located downstream (in the 3’ direction) from the
promoter so that if repressor is bound RNA Polymerase can’t transcribe
©2000 Timothy G. Standish
The The LacLac Operon: Operon:When Glucose Is Present But Not LactoseWhen Glucose Is Present But Not Lactose
Repressor Promoter LacY LacALacZOperatorCAPBinding
RNAPol.
Repressor
Repressor
Repressor mRNA
Hey man, I’m constitutive
Come on, let me through
No wayJose!
CAP
©2000 Timothy G. Standish
The The LacLac Operon: Operon:When Glucose And Lactose Are PresentWhen Glucose And Lactose Are Present
Repressor Promoter LacY LacALacZOperatorCAPBinding
Repressor
Repressor mRNA
Hey man, I’m constitutive
CAP
Lac
Repressor
Repressor
X
RNAPol.
RNAPol.
Great, I can transcribe!
Some transcription occurs, but at a slow rate
This lactose has bent me
out of shape
©2000 Timothy G. Standish
The The LacLac Operon: Operon:When Lactose Is Present But Not GlucoseWhen Lactose Is Present But Not Glucose
Repressor Promoter LacY LacALacZOperatorCAPBinding
Repressor
Repressor mRNA
Hey man, I’m constitutive
CAPcAMP
Lac
Repressor
Repressor
X
This lactose has bent me
out of shape
CAPcAMP
CAPcAMP
Bind to mePolymerase
RNAPol.
RNAPol.
Yipee…!
©2000 Timothy G. Standish
The The LacLac Operon: Operon:When Neither Lactose Nor Glucose Is PresentWhen Neither Lactose Nor Glucose Is Present
Repressor Promoter LacY LacALacZOperatorCAPBinding
CAPcAMP
CAPcAMP
CAPcAMP
Bind to mePolymerase
RNAPol.
Repressor
Repressor mRNA
Hey man, I’m constitutive
Repressor
STOPRight therePolymerase
Alright, I’m off to the races . . .
Come on, let me through!
©2000 Timothy G. Standish
The The TrpTrp Operon Operon Genes in the trp operon allow E. coli bacteria to make
the amino acid tryptophan Trp operon genes encode enzymes involved in the
biochemical pathway that converts the precursor chorismate to tryptophan.
The trp operon is controlled in two ways:– Using a repressor that works in exactly the opposite way
from the lac operon repressor– Using a special attenuator sequence
©2000 Timothy G. Standish
The TryptophanThe TryptophanBiochemical PathwayBiochemical Pathway
O
-OOC
OH
HN
HH
-2O3P
OH
HH
CH2O
5-Phosphoribosyl--Pyrophosphate PPi
N-(5’-Phosphoribosyl)-anthranilate
COO-
COO-
H
CH2
C
HO
HO
Chorismate
-OOCOH-2O3PO CH2
NH
CH
C C
H
OH
C
H
OH
Enol-1-o-Carboxyphenylamino-1-deoxyribulose phosphate
NH
-OOC CH2
NH3+
C
H
Tryptophan
H2OSerine
Anthranilate
COO-
NH2
Glutamate +Pyruvate
Glutamine
CO2+H2O -2O3PO CH2
CH
C C
H
OH
C
H
OH
NH
Indole-3-glycerol phosphateGlyceraldehyde-3-phosphate
NH
Indole
Anthranilate synthetase(trpE and D)
Anthranilate synthetase
N-(5’-Phosphoribosyl)-anthranilateisomerase Indole-3’-glycerol phosphate synthetase (trpC)
Tryptophan synthetase(trpB and A)
N-(5’-Phosphoribosyl)-Anthranilate isomerase Indole-3’-glycerol phosphate synthetase
Tryptophan synthetase
©2000 Timothy G. Standish
The The TrpTrp Operon: Operon:When Tryptophan Is PresentWhen Tryptophan Is Present
STOPRight therePolymerase
Trp
Trp
Repressor
Repressor
Repressor Promo. trpD trpBLead.Operator trpAtrpCtrpEAten.RNAPol.
FoiledAgain!
Repressor mRNA
Hey man, I’m constitutive
©2000 Timothy G. Standish
The The TrpTrp Operon: Operon:When Tryptophan Is AbsentWhen Tryptophan Is Absent
Repressor
Repressor Promo. trpD trpBLead.Operator trpAtrpCtrpEAten.
Repressor mRNA
Hey man, I’m constitutive
RNAPol.
RNAPol.
Repressor needs hislittle buddy tryptophan if
I’m to be stoppedI need
tryptophan
©2000 Timothy G. Standish
AttenuationAttenuationThe trp operon is controlled both by a repressor and
attenuationAttenuation is a mechanism that works only because
of the way transcription and translation are coupled in prokaryotes
Therefore, to understand attenuation, it is first necessary to understand transcription and translation in prokaryotes
©2000 Timothy G. Standish
3’
5’
5’
3’
Transcription And Translation Transcription And Translation In ProkaryotesIn Prokaryotes
Ribosome
Ribosome5’
mRNA
RNAPol.
©2000 Timothy G. Standish
Met-Lys-Ala-Ile-Phe-Val-AAGUUCACGUAAAAAGGGUAUCGACA-AUG-AAA-GCA-AUU-UUC-GUA-
Leu-Lys-Gly-Trp-Trp-Arg-Thr-Ser-STOPCUG-AAA-GGU-UGG-UGG-CGC-ACU-UCC-UGA-AACGGGCAGUGUAUU
CACCAUGCGUAAAGCAAUCAGAUACCCAGCCCGCCUAAUGAGCGGGCUUUU
Met-Gln-Thr-Gln-Lys-ProUUUU-GAACAAAAUUAGAGAAUAACA-AUG-CAA-ACA-CAA-AAA-CCG trpE . . .Terminator
The The TrpTrp Leader and Leader and AttenuatorAttenuator
4
1 2
3
©2000 Timothy G. Standish
The mRNA Sequence Can The mRNA Sequence Can Fold In Two WaysFold In Two Ways
4
1 23
Terminatorhairpin
4
1 2
3
©2000 Timothy G. Standish
3’
5’
5’
3’
The Attenuator The Attenuator When Starved For TryptophanWhen Starved For Tryptophan
41
23
RNAPol.
Ribosome
Leader peptide
Ribosome stalls over sequence 1 and sequence 2 binds to sequence 3 preventing formation of the 3, 4 hairpin
Help,I need
Tryptophan
©2000 Timothy G. Standish
3’
5’
5’
3’
The Attenuator The Attenuator When Tryptophan Is PresentWhen Tryptophan Is Present
4
1 2
3
RNAPol.
Ribosome
Leader peptide is released
Ribosome passes over sequence 1 and onto sequence 2 allowing sequence 3 to form the 3, 4 hairpin
©2000 Timothy G. Standish
3’
5’
5’
3’
The Attenuator The Attenuator When Tryptophan Is PresentWhen Tryptophan Is Present
RNAPol.
Ribosome
The 3, 4 hairpin destabilizes the elongation complex
4
1 2
3
©2000 Timothy G. Standish
3’
5’
5’
3’
The Attenuator The Attenuator When Tryptophan Is PresentWhen Tryptophan Is Present
Ribosome
RNAPol.
RNA polymerase falls off ending transcription
4
1 2
3
©2000 Timothy G. Standish