Gene Expression/transcription(e Coli) Flashcards
Why do we study gene expressiom regulation in E. coli
Key pathogens can provide insight to how to target them via antibiotics (targets expression)
What strand is used to produce mrna
Anti sense strand (template)
Antisense means opposite to mrna
What does rna polymerase need in terms of ions to catalyse phosphodiester bonds
Mg2+
What are the precursors added to make mrna
Ribonucleotide triphosphates
RATP rUTP rGTP rCTP
What 2 consensus sequences are recognised by a holoenzyme at the promoter region
-35 and at -10
Negative because start site is +1
Why are consensus sequences called hexameric
6 bases each
What does strength of a promoter depend on
Depends on how close the bases on a promoter region are to being the same as the consensus sequence
(If basically identical = strong promoter = more transcription)
What is rna polymerase made of (subunits)
2 x alpha
1 x beta
1 x beta’
1 x w (omega)
Added sigma factor to produce a holoenzyme
Why is the production of a holoenzyme needed
Sigma is needed to direct recognition of promoter -35 and -10 and rna polymerase can bind
What gene is sigma 70 produced by
rPON gene
Why are there alternate sigma factors to sigma 70 and give example
To match the cellular environment
For example sigma H is for heat shock genes in response to heat shock of a bacterium
What is the complex of the holoenzyme and dna called in INITIATION and what property does it have
Closed complex - unstable
Open complex occurs in elongation. What happens
Sigma factor opens DNA forming a transcription bubble at the +1 site
Rna starts to incorporate rNTPS
When is sigma factor released
In elongation when the transcription bubble moves along to form 5’ to 3’ mrna
How does elongation occur
Catalysis of phosphodiester bonds growing 5’ to 3’ between rNTPS depending on bad pair with template strand
PPi (pyrophosphate is lost)
What other technique is used instead of proofreading by exonucleases like dna pol to try and reduce error rate in base pairs
Backtracking - breaking previous base pairs (not favoured)
What 2 types of terminations is there?
Rho factor independent
Rho factor dependant
What bases makes up factor independant terminator/termination of transcription
AT base pairs following GC palindromic sequences
What shape does mrna produce due to rho independent pallindromic sequence
It forms a stem loop shape (via G-C base pairs)
Mrna forms BP with itself
How does folding of mrna into stem loop cause weak interaction with dna allowing mrna to detach?
Only A U base paired with dna which is week and causes mrna to detach
Why does polymerase detach from dna in rho independent termination
It becomes unstable on the helixes when mrna detaches
What is rho factor made up of and what is its job
6 helical subunits
It unwinds rna-dna or rna-rna interaction
What is unwinding by rho factor dependant on
ATP hydrolysis
What occurs in rho dependant termination
RHo factor loads into mrna at the C rich sites
Terminator sequence stops mrna transcription
Rho then unwinds the mrna from dna via atp hydrolysis
What is RUT site ?
Rho utilisation site
C rich region on mrna helping it bind
What 2 ways is gene regulation response to change in cellular environments
1- need different genes expressed
2- different level of genes expressed needed
What is the repression strategy
Repressor molecule has a negative effect on expression
It stops rna polymerase binding at the -35 -10 promoter sequence
= no transcription
How does activation strategy work
A protein activator is needed to bind to rna polymerase to help it bind to dna
Why do protein activators be needed for rna pol to bind
Because it is a weak promoter sequence = rna not able to bind efficiently
What happens in terms of rna polymerase when promoter sequences are ‘weak’ - ie not close to consensus
They are not recognised easily
What is the lac operon (example of gene regulation)
A cluster of genes which are under control of a single promoter
Name the components of the lac operon including what enzymes they code for
Plac - promoter region
Lac operator (start site +1)
Lac z - b galactosidase
Lac Y - permease
Lac A- acetylase
WhT do the cluster genes on lac operon help ecoli to do
Allow use of lactose for carbon source instead of glucose
What gene produces the lac repressor which inhibits lac ZYA expression
PlacI gene
Explain the structure of the lac repressor and the 3 domains it has
Homotetramer
Dna binding domain
Regulatory domain
Homotetramisation domain
What does the homotetramer lac repressor bind to which stops gene expression of ZYA? By stopping rna recognition
The lac operator (start site +1)
What does the lac operator consist of in its sequence and does it bind with high affinity to lac repressor?
It’s a palindrome sequence
It binds to lac depressor tightly
When is the transcription of the lac operon genes initiated?
When lactose is present
When glucose isn’t present (would be preferred c source)
What binds to the lac repressor which lowers its affinity to the lac operator site?
Allolactose (only present in presence of lactose)
Explain the way lactose going into cell produces allolactose which can then start gene expression
Lactose enters via galactose permease
The b galactosidase then turns it into allolactose
(Or it can be hydrolysed into galactose and glucose)
Is transcription fully blocked by the lac repressor
No there is still a small amount of transcription of ZYA genes
How does glucose at low levels influence gene expression
Glucose at low levels is not enough to inhibit adenylate cyclase
More cAMP is produced
How does cAMP presence in low glucose levels cause gene expression
Camp binds to catabolite activator protein
This then binds to rna polymerase and helps it recognise and bind to the weak promoter sequence
Name the lac activator protein bound to cAMP
Catabolite activator protein CAP
Why is both the activator (low glucose) and allolactose (high lactose presence) needed for gene expression
Allolactose is only enough to remove the repressor, but the rna polymerase needs the CAP protein bound with cAMP to be able to recognise the weak promoter sequence and initiate transcription
What is the difference between allolactose and lactose
The alpha glycosidic bond is at 1-6’ instead of 1-4
How does allolactose change the shape of the lac repressor lacI
Seperates the dna binding domains
= lower affinity for the lac operator 1
