©1999 Timothy G. Standish
Hebrews 1:1-2
1 God, who at sundry times and in divers manners spake in time past unto the fathers by the prophets,
2 Hath in these last days spoken unto us by his Son, whom he hath appointed heir of all things, by whom also he made the worlds;
©1999 Timothy G. Standish
Controlling Controlling Gene ExpressionGene Expression
Timothy G. Standish, Ph. D.
©1999 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 genes are called constitutive genes and are expressed by all cells.
Other genes are only needed by certain cells or at specific times. The expression of these inducible genes is tightly controlled in most cells.
For example, beta cells in the pancreas make the protein insulin by expressing the insulin gene. If neurons expressed insulin, problems would result.
©1999 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
©1999 Timothy G. Standish
The The LacLac Operon Operon Genes in the lac operon allow E. coli bacteria to
metabolize lactose Lactose is a sugar that E. coli is unlikely to encounter, 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
©1999 Timothy G. Standish
The The LacLac Operon - Parts Operon - Parts The lac operon is made up of a control region and four genes The four genes are:
– LacZ - -galactosidase - An enzyme that hydrolyzes the bond between galactose and glucose
– LacY - Codes for a permease that lets lactose across the cell membrane
– LacA - Transacetylase - An enzyme whose function in lactose metabolism is uncertain
– Repressor - A protein that works with the control region to control expression of the operon
©1999 Timothy G. Standish
The The LacLac Operon - Control Operon - Control The control region is made up of two parts: Promoter
– These are specific DNA sequences to which RNA
Polymerase binds so that transcription can occur– The lac operon promoter also has a binding site for another protein
called CAP 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
©1999 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
©1999 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
©1999 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…!
©1999 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!
©1999 Timothy G. Standish
The The TrpTrp Operon Operon Genes in the trp operon allow E. coli bacteria to
make the amino acid tryptophan Enzymes encoded by genes in the trp operon are all
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
©1999 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
©1999 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
©1999 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
©1999 Timothy G. Standish
AttenuationAttenuation
The trp operon is controlled both by a repressor and attenuation
Attenuation 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
©1999 Timothy G. Standish
3’
5’
5’
3’
Transcription And Translation Transcription And Translation In ProkaryotesIn Prokaryotes
Ribosome
Ribosome5’
mRNA
RNAPol.
©1999 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
©1999 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
©1999 Timothy G. Standish
3’
5’
5’
3’
The Attenuator The Attenuator When Starved For TryptophanWhen Starved For Tryptophan
41
23
RNAPol.
Ribosome
Help,I need
Tryptophan
©1999 Timothy G. Standish
3’
5’
5’
3’
The Attenuator The Attenuator When Tryptophan Is PresentWhen Tryptophan Is Present
41
23
RNAPol.
Ribosome
RNAPol.
©1999 Timothy G. Standish
Control Of Expression In Control Of Expression In EukaryotesEukaryotes
Some of the general methods used to control expression in prokaryotes are used in eukaryotes, but nothing resembling operons is known
Eukaryotic genes are controlled individually and each gene has specific control sequences preceding the transcription start site
In addition to controlling transcription, there are additional ways in which expression can be controlled in eukaryotes
©1999 Timothy G. Standish
Eukaryotes Have Large Eukaryotes Have Large Complex GenomesComplex Genomes
The human genome is about 3 x 109 base pairs or ≈ 1 m of DNA
Because humans are diploid, each nucleus contains 6.3 x 109 base pairs or ≈ 2 m of DNA
That is a lot to pack into a little nucleus!
©1999 Timothy G. Standish
Eukaryotic DNA Must be Eukaryotic DNA Must be PackagedPackaged
Eukaryotic DNA exhibits many levels of packaging
The fundamental unit is the nucleosome, DNA wound around histone proteins
Nucleosomes arrange themselves together to form higher and higher levels of packaging.
©1999 Timothy G. Standish
Highly Packaged DNA Cannot Highly Packaged DNA Cannot be Expressedbe Expressed
The most highly packaged form of DNA is “heterochromatin”
Heterochromatin cannot be transcribed, therefore expression of genes is prevented
Chromosome puffs on some insect chomosomes illustrate where active gene expression is going on
©1999 Timothy G. Standish
Only a Subset of Genes is Only a Subset of Genes is Expressed at any Given TimeExpressed at any Given Time
It takes lots of energy to express genes Thus it would be wasteful to express all genes all the time By differential expression of genes, cells can respond to
changes in the environment Differential expression, allows cells to specialize in
multicelled organisms. Differential expression also allows organisms to develop
over time.
©1999 Timothy G. Standish
A “Simple” Eukaryotic GeneA “Simple” Eukaryotic Gene
Terminator Sequence
Promoter/Control Region
Transcription Start Site
3’5’
RNA Transcript
Introns
Exon 2 Exon 3Int. 2Exon 1 Int. 1
3’ Untranslated Region5’ Untranslated Region
Exons
©1999 Timothy G. Standish
Eukaryotic mRNAEukaryotic mRNA
Protein Coding Region
3’ Untranslated Region5’ Untranslated Region
Exon 2 Exon 3Exon 1 AAAAAG 3’5’
3’ Poly A Tail5’ Cap
RNA processing achieves three things: Removal of introns Addition of a 5’ cap Addition of a 3’ tail
This signals the mRNA is ready to move out of the nucleus and may control its lifespan in the cytoplasm
©1999 Timothy G. Standish
5’DNA
3’
EnhancersEnhancers
Enhancer Transcribed Region
3’5’ TF TFTF
3’5’ TF TFTF
5’RNA
RNAPol.
RNAPol.
Many bases
Promoter