1. Regulation of gene expression : transcription in prokaryotic cells

Molecular Biology for prokaryotic cells

1.1 General structure of an operon

Promoter, +1 = Transcription Starting Site (TSS), Shine Dalgardo (SD) sequence and a Terminator

1.1 General structure of a Promoter

2 consensus sequences :

• Hexamer - 35

• Pribnow box -10

1.1 ARN pol structure

5 sub units : 

• X2 α

• β

• β’

• ω

= Core enzyme

1.1 Role and structure of α

2 extremities αCTD & αNTD

• αNTD —> associates to β

• αCTD —> interacts w/ DNA 7 transcription regulatory proteins

1.1 Role of β & β’ sub-units

contain active site to remain attached to DNA 

non specific binding to DNA strand 

1.1 Function of ω sub-unit

Facilitates assembly of RNA polymerase 

1.1 Why does ARN binds w/ sigma σ sub-unit ? What is the complex called ? 

To initiate the transcription as σ sub-unit is responsible for recognising the consensus sequences on the promoter 

Core enzyme + σ = Holoenzyme (6 sub-units)

1.1 Specificity and function of sigma σ sub-unit

contains many sub-regions that’s have a high specificity for the promoter and their consensus sequences

prevent transcription starting from anywhere 

1.1 How can a promoter be a strong promoter

Hexamer & Prinbow box have a sequence identical/very similar to consensus sequence

Strong binding between domains of σ sub-unit with the sequences

Strong association of RNA pol w/ promoter 

• initiation of transcription 

1.1 What makes a weak promoter

Hexamer & Prinbow box sequences are different from consensus sequences

Weak binding between domains of σ sub-unit

Weak association of RNA pol w/ promoter 

• Dissociation of ARN pol from promoter —> no transcription 

1.1 E.g. of σ sub-unit, and the domains that bind to Hexamer & Prinbow box on the promoter in E.coli

σ70 = σ sub-unit

2 domains ": 

• 4.2 binds to Hexamer

• 2.4 binds to Prinbow box

1.1 What are the 3 steps of INITIATION of transcription ? 

1. Fixation of ARN pol (holoenzyme) on the promoter = closed complex (double strand of DNA remains closed)

2. Double strands of DNA are separated = open complex

3. Promoter escape (holoenzyme —> core enzyme)

1.1 What is the closed complex in the first step of INITIATION of transcription 

RNA pol/Holoenzyme is associated to the promoter of the DNA 

DNA double strand remains closed 

1.1 Explain what happens in the 2nd step of INITIATION (open complex) of transcription

DNA is opened by 13bp at the Prinbow box ( as only 2 H bonds between T-A compared 3 in C-G) by the Holoenzyme

Transcription blocked by RNA Pol/Holoenzyme at an RNA size of ≤ 9 nucleotides as

1.1 Explain promoter escape (3rd step in INITIATION of transcription)

• After 10bp have been transcribes the σ70 sub-unit dissociated from core enzyme 

• RNA pol/Core enzyme is no longer fixated to the promoter 

• RNA pol/Core enzyme moves down DNA and Strats elongation w/ complementary base pairing

1.2 Repressor general model to control the promoter ; location in between -35 & -10  and function

Repressor binding site in between -35 et -10

Active repressor : 

• Repressor protein binds to REP binding site = active repressor 

• Inhibition of binding of RNA pol/holoenzyme 

• No transcription

Inactive repressor :

• REP protein doesn’t bind to REP binding site due to a signal

• Holoenzyme can bind to the promoter

• Initiate transcription 

1.2 Repressor general model to control the promoter ; location after -10  and function

Rep binding site further down after -10

Active repressor :

• REP protein binds to REP binding site 

• Holoenzyme can bind to the promoter but is blocked by Rep protein 

• Holoenzyme can’t move forward 

• No transcription

Inactive repressor : 

• Rep doesn’t bind to REP binding site due to signal

• No blockage from REP protein

• Holoenzyme not blocked and move forward 

• Transcription occurs 

1.2 General activator model to control the promoter ; Fixation of weak promoter

Activator binding site before -35

Inactive activator 

• ACT protein doesn’t bind to ACT binding site

• Holoenzyme binds but quickly dissociates from DNA

• No transcription

Active activator

• ACT binds to ACT binding site due to a signal

• Stabilises intégration of holoenzyme w/ the promoter

• Transcription takes place

1.2 General activator model to control the promoter ; Transition from close —> open complex for a weak promoter

ACT binding site before -35

Inactive Activator 

• ACT doesn’t bind to ACT binding site

• Holoenzyme is bound to promoter

• Unable to open double stranded DNA —> remains in closed complexe

• No transcription

Active Activator

• ACT binds to ACT binding site 

• Allows Holoenzyme to enter the open complex 

• DNA is opened

• Transcription takes place 

1.2 Class of Activators

Class I : fixation of holoenzyme

Class II : closed —> open complex 

1.2 E.g of Class I Activators

 fixation around -60

interaction of αCTD with ACT protein to stabilise interacation of holoenzyme w/ promoter

1.2 E.g of Class II Activator close to holoenzyme

= The majority of ACT

fixation around -40 

ACT interacts w/ αCTD, αNTD & σ sub units 

stabilises interaction of holoenzyme w promoter 

allows transition from closed to open complex 

1.2 E.g of Class II Activator far from holoenzyme

ACT binding site is distant from Holoenzyme

Bending protein blinds to its binding site before the ACT binding site —> DNA bends

ACT protein bound to its binding site can now interact w/ holenzme

1.3.1 Operon tryptophan

Inactive repressor Trp R : absence of Trp

• transcription of REP

• no binding of REP w/ Trp

• no binding to operator —> no transcription of enzymes needed for biosynthesis of Trp

Active repressor TrpR : presence of Trp

• Trp binds to REP

• complex binds to operator so holoenzyme can’t bind to the promoter

• no transcription

1.3.1 Trp R structure

1. dimeric

2. di-α helix

3. bound w/ Trp —> TrpR can bind w/ major groove of DNA ( conformational change allows this)

1.3.1 Can TrpR repress other genes ?

YES

• Trp (70 times)

• aroH (2 times) ; genes involved in synthesis of aromatic AA

• TrpR (3 times) ; self regulation 

1.3.1 How is there a differences in repression efficiency between the same repressor and different genes ? 

TrpR has a different affinity for the operators of each gene (less similarity —> weaker fixation —> less repression)

The position of the operator on each gene : stronger when operator is around -10 and weaker around -35)

Depends on the strength of the promoter : if strong promoter —> strong competition between TrpR & holoenzyme —> less repression [and vise versa]

1.3.2 Principal of repression by blocking the escaping of the holoenzyme (step 3 of initiation of transcription) e.g protein p4 in bacteriophage 

1. bacteriophage injects genome in bacteria 

2. viral cycle : expression of “early” genes first but no expression of the “late” genes 

3. expression of “late” genes later but no “early” genes

1.3.2 What is a p4 protein and where is the p4 binding sites located on the DNA?

p4 = “early” gene protein

binding site located inform of promoter of the “early” and “late” genes

1.3.2 Expression of “early” genes in bacteriophage using p4

At the beginning : 

promoter for “early” genes = strong

• strong binding between holoenzyme & promoter of “early” genes e.g. p4

• strong expression of p4

promoter for “late” genes = weak

• no transcription of “late” genes at the moment

1.3.2 Expression of “late” genes in bacteriophage using p4

After expression of “early genes” : 

promoter for “early” genes = strong 

p4 binds to its binding site infront of its promoter for “early” genes 

blocks the holoenzyme from escaping as the binding of the holoenzyme w/the promoteur is very strong 

no transcription of “early” genes

promoter for “late” genes = weak

p4 binds to its binding site infant of the promoter for “late”genes

stabilises the fixation of there holoenzyme

transcription of “late” genes

1.3.2 what is p4 to “early” and “late” genes

to “early” genes p4= repressor

to “late” genes p4 = activator

1.3.3. lactose operon ; how many operators (binding sites for lacI) does it contain and genes does it code for

2 operators ; O1 & O2

3 sequences : 

1. lac z —> beta-galactosidase 

2. lac y —> perméase

3. lac A —> transacetylase

1.3.3 is the promoter for lactose operon weak or strong ?

weak

1.3.3. allolactose role in regulating initiation & LacI

1. Lactose enters the bacteria via permease

2. lactose is transformed into allolactose and galactose + glucose by β-Gal

3. allolactose = ligand that binds to LacI REP —> inactivating LacI

4. allolactose = inductor of lactose operon

1.3.3 by what is the lactose operon regulated by ?

LacI will repress the transcription in the absence of lactose 

CAP will activate the transcription when little glucose is available 

1.3.3 what is the CAP protein

Class I activator ( stabilises interaction of holoenzyme w/ promoter )

1.3.4 how does the structure of LacI REP change in presence of allolactose

LacI forms a homodimer = active form and can bind to DNA to form a tetramer

When allolactose present it binds to to the homodimer of LacI = change of conformation inactivates the REP & can no longer bind to DNA

1.3.4 How is CAP regulated ?

Lots of Glc outside of the bacteria —> inactive CAP

• Glc in imported by a transporter 

• Glc is phosphorylated so cannot exit the bacteria 

• cyclic adenylate transforms ATP to cAMP (very little)

No Glc outside of the bacteria —> active CAP

• no transcription of Lactose operon

• cyclic adenylate is phosphorylated and transforms lots of ATP into cAMP

• cAMP binds to CAP dimer —> active form

• CAP binds to activator domain of the promoter

• transcription of lactose operon

Glc already present —> no need for lactose

No Glc present —> lactose needed to be metabolised into Glc 

1.3.4 Is lactose operon transcribed in the absence of lactose 

NO 

LacI = active and can bind to operator site 1 —> blocking holoenzyme from binding to promoter

LacI binds to operator site 2 —> increasing repression as there’s complete blockage

—> no transcription

1.3.4 Is lactose operon transcribed in presence of lactose & glucose

YES but very little 

lactose —> allolactose which binds to LacI —> inactivating REP LacI —> holoenzyme can bind to WEAK promoter -35 & -10 

CAP inactived by Glc —> so weak interaction between holoenzyme and promoter 

—> little to no transcription 

1.3.4 Is lactose operon transcribed in presence of lactose & absence of glucose

YES

lactose —> allolactose which binds to LacI REP —> no inhibition of transcription 

cyclic adenylate phosphorylated —> strong production of cAMP —> cAMP binds to CAP dimer activating it —> CAP binds to activator binding site stabilising holoenzyme-promoter interaction 

—> good transcription 

1.3.4 so what is an example of a class one activator couples to a repressor ?

lactose operon 

activated by CAP = Class I activator 

inhibited by LacI = REP

1.3.5 what 2 regulators are an example of a class II activator (closed —> open complex)

1. luxR regulates the expression of lux operon

2. FadR regulates the expression of fabA and fabL genes

1.3.5 explain how LuxR regulates lux operon 

free bacteria in water : lux operon is off —> no bioluminescence

bacteria colonising squid : luc operon on —> transcription of enzymes for luciferase —> bioluminescence  

1.3.5 what is the mecanism of luxR when the bacteria is free in the water

• bacteria produces small amount of AHL (=Acyl homoserine lactose a ligand for luxR)

• AHL diffuses out of the bacteria and dilutes in the water 

• little AHL present in bacteria —> inactive LuxR —> no transcription of enzymes for luciferase 

—> no bioluminescence

1.3.5 what is the mecanism of luxR when the bacteria has colonised a squid

• bacteria is in a closed compartment/specialised organ

• AHL produced by bacteria remains in the bacteria so high [AHL] in bacteria

• AHL binds to luxR —> dimer formed

• luxR binds to activator site -42 allowing stabilisation of holoenzyme and for it to go from closed to open complex 

—> bioluminescence

1.3.5 what are the genes fabA and fadL involved in and what regulates them ?

fabA = Fatty Acid Biosynthesis : Fatty Acid (FA) synthesis for membrane lipids

• this is expressed when there are no exogenous/exterior FA’s

fadL = Fatty Acid Degradation : FA Brocken down to be used for membrane lipids or into acetyl-CoA

• this is expressed when there are exogenous FA’s

Both ones are regulated by FadR protein

1.3.5 how is FadR activated

active form = unbound from FA

inactive form = bound to FA 

1.3.5 what strength are the primers of fabA and fadL 

weak : fabA

strong : fadL

1.3.5 what happens to the expression of fabA and fadL when there is presence of FA

exogenous FA present 

fabA :

FA binds to fadR —> inactive form 

cannot bind to fadR binding site in-front of the weak fabA promoter 

no fixation of holoenzyme 

—> no transcription —> no FA biosynthesis

fadL : 

FA binds ro fadR —> inactive form  

doesn’t bind to fadR binding site in-front of the strong fadL promoter 

holoenzyme binds to promoter 

—> transcription —> FA degradation

1.3.5 what happens to the expression of fabA and fadL in absence of FA

fabA :

no binding of FA to fadR —> active form 

FadR binds to FadR binding site infant of the weak promoter of fabA

stabilises holoenzyme-promoter interaction & promotes transition from closed to open complex 

—> transcription —> FA biosynthesis 

fadL : 

FadR = active as no FA bound to it 

FadR binds to 2 operators located between -35 & -10 of the strong promoter of fadL

steric hindrance ; prevents holoenzyme from binding to -35 & -10

—> no transcription —> no FA degradation

1.3.6 what is an examples of the use of 2 activators working together?

respiration : nitrate & fumarate reductase operon

1.3.6 how does anaerobic respiration work ?

when O2 is absent E.coli uses other e- acceptors like :

• nitrate NO3- —> produces less ATP than O2

• fumarate —> produces less ATP than NO3-

use of nitrate and fumarate requires enzymes : nitrate and fumarate reductase