ecclesiastes 3:1

©2000 Timothy G. Standish

Ecclesiastes 3:11 To every thing there is a

season, and a time to every purpose under the heaven:


Initiation of Initiation of TranscriptionTranscription

Timothy G. Standish, Ph. D.


All Genes Can’t be Expressed All Genes Can’t be Expressed At The Same TimeAt The Same Time

Some gene products are needed by all cells all the time. These constitutive genes are expressed by all cells.

Other genes are only needed by certain cells or at specific times, expression of these inducible genes is tightly controlled in most cells.

For example, pancreatic cells make insulin by expressing the insulin gene. If neurons expressed insulin, problems would result.


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


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.

Production of lactose metabolizing enzymes when not needed would be wasteful

Metabolizing lactose for energy only makes sense when two criteria are met:1 Other more readily metabolized sugar (glucose) is unavailable2 Lactose is available


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 - Hydrolizes 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


The The LacLac Operon - Control Operon - Control The control region is made up of two parts:1 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

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


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


The The LacLac Operon: Operon:When Glucose And Lactose Are PresentWhen Glucose And Lactose Are Present


Repressor

Repressor mRNA


CAP

Lac

Repressor

Repressor

XRNAPol.

RNAPol.

Great, I can transcribe!

Some transcription occurs, but at a slow rate

This lactose has bent me

out of shape


The The LacLac Operon: Operon:When Lactose Is Present But Not GlucoseWhen Lactose Is Present But Not Glucose


Repressor

Repressor mRNA


CAPcAMP

Lac

Repressor

Repressor

X

This lactose has bent me

out of shape

CAPcAMP

CAPcAMP

Bind to mePolymerase

RNAPol.

RNAPol.

Yipee…!


The The LacLac Operon: Operon:When Neither Lactose Nor Glucose Is PresentWhen Neither Lactose Nor Glucose Is Present


CAPcAMP

CAPcAMP

CAPcAMP

Bind to mePolymerase

RNAPol.

Repressor

Repressor mRNA


Repressor

STOPRight therePolymerase

Alright, I’m off to the races . . .

Come on, let me through!


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


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

Antrhanilate

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


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



The The TrpTrp Operon: Operon:When Tryptophan Is AbsentWhen Tryptophan Is Absent

Repressor

Repressor Promo. trpD trpBLead.Operator trpAtrpCtrpEAten.

Repressor mRNA


RNAPol.

RNAPol.

Repressor needs hislittle buddy tryptophan if

I’m to be stoppedI need tryptophan


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


3’

5’

5’

3’

Transcription And Translation Transcription And Translation In ProkaryotesIn Prokaryotes

Ribosome

Ribosome5’

mRNA

RNAPol.


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 Trp Leader and The Trp Leader and AttenuatorAttenuator

4

1 2

3


The mRNA Sequence Can The mRNA Sequence Can Fold In Two WaysFold In Two Ways

4

1 23

Terminatorhairpin

4

1 2

3


3’

5’

5’

3’

The Attenuator The Attenuator When Starved For TryptophanWhen Starved For Tryptophan

41

23

RNAPol.

Ribosome

Help,I need

Tryptophan


3’

5’

5’

3’

The Attenuator The Attenuator When Tryptophan Is PresentWhen Tryptophan Is Present

41

23

RNAPol.

Ribosome

RNAPol.


Expression Control In EukaryotesExpression Control In Eukaryotes 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


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 x 109 base pairs or ≈ 2 m of DNA

Some gene families are located close to one another on the same chromosome

Genes with related functions appear to be distributed almost at random throughout the the genome


Highly Packaged DNA Cannot Highly Packaged DNA Cannot be Expressedbe Expressed

Because of its size, eukaryotic DNA must be packaged

Heterochromatin, the most highly packaged form of DNA, cannot be transcribed; therefore expression of genes is prevented

Chromosome puffs on some insect chomosomes illustrate areas of active gene expression


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.


DNA

Cytoplasm

NucleusG AAAAAA

Export

Degradation etc.G AAAAAA

Control of Gene ExpressionControl of Gene Expression

G AAAAAA

RNAProcessing

mRNA

RNA

Transcription

Nuclear pores

Ribosome

Translation

Packaging

ModificationTransportation

Degradation


Logical Expression Control PointsLogical Expression Control Points DNA packaging Transcription RNA processing mRNA Export mRNA masking/unmasking and/or

modification mRNA degradation Translation Protein modification Protein transport Protein degradation

Increasing cost

The logical place to control

expression is before the

gene is transcribed


Three Eukaryotic Three Eukaryotic RNA PolymerasesRNA Polymerases

1RNA Polymerase I - Produces rRNA in the nucleolus, accounts for 50 - 70 % of transcription

2RNA Polymerase II - Produces mRNA in the nucleoplasm - 20 - 40 % of transcription

3RNA Polymerase III - Produces tRNA in the nucleoplasm - 10 % of transcription


A “Simple” Eukaryotic GeneA “Simple” Eukaryotic Gene

Terminator Sequence

Promoter/Control Region

Transcription Start Site 5’ Untranslated Region 3’ Untranslated Region

Exons

Introns

3’5’ Exon 2 Exon 3Int. 2Exon 1 Int. 1

RNA Transcript


5’DNA

3’

EnhancersEnhancers

Enhancer Transcribed Region

3’5’ TF TFTF

3’5’ TF TFTF

5’ RNA

RNAPol.

RNAPol.

Many bases

Promoter


Eukaryotic RNA Polymerase IIEukaryotic RNA Polymerase II RNA polymerase is a very fancy enzyme that

does many tasks in conjunction with other proteins

RNA polymerase II is a protein complex of over 500 kD with more than 10 subunits:


Eukaryotic RNA Polymerase II Eukaryotic RNA Polymerase II PromotersPromoters

Several sequence elements spread over about 200 bp upstream from the transcription start site make up RNA Pol II promoters

Enhancers, in addition to promoters, influence the expression of genes

Eukaryotic expression control involves many more factors than control in prokaryotes

This allows much finer control of gene expression


RNA Pol. II

InitiationInitiation

T. F.

RNA Pol. II

5’mRNA

Promoter

T. F.

T. F.


Eukaryotic PromotersEukaryotic Promoters

5’ Exon 1Promoter

Sequence elements

~200 bp

TATA

~-25

Initiator“TATA Box”

Transcription start site

(Template strand) -1+1SSTATAAAASSSSSNNNNNNNNNNNNNNNNNYYCAYYYYYNN

S = C or G Y = C or T N = A, T, G or C


InitiationInitiationTFIID BindingTFIID Binding

-1+1


TFIID

“TATA Box”

TBP Associated Factors (TAFs)

TATA Binding Protein (TBP)


InitiationInitiationTFIID BindingTFIID Binding

TFIID

80o Bend-1+1



InitiationInitiationTFIIA and B BindingTFIIA and B Binding

TFIID

TFIIA

-1+1


TFIIB


InitiationInitiationTFIIF and RNA Polymerase BindingTFIIF and RNA Polymerase Binding

TFIID

TFIIA

-1+1


TFIIB

RNA PolymeraseTFIIF


InitiationInitiationTFIIE BindingTFIIE Binding

TFIID

TFIIA

-1+1


RNA PolymeraseTFIIBTFIIF

TFIIE

TFIIE has some helicase activity and may by involved in unwinding DNA so that transcription can start


InitiationInitiationTFIIH and TFIIJ BindingTFIIH and TFIIJ Binding

TFIID

TFIIA

-1+1



TFIIE

TFIIH has some helicase activity and may by involved in unwinding DNA so that transcription can start

TFIIH

P PP

TFIIJ



TFIID

TFIIA

-1+1



TFIIETFIIH

P PP

TFIIJ



-1+1


RNA PolymeraseP P

P

ecclesiastes 3:1

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