Gene Expression/transcription(e Coli)

Question 1

Why do we study gene expressiom regulation in E. coli

Answer 1

Key pathogens can provide insight to how to target them via antibiotics (targets expression)

Question 2

What strand is used to produce mrna

Answer 2

Anti sense strand (template)

Antisense means opposite to mrna

Question 3

What does rna polymerase need in terms of ions to catalyse phosphodiester bonds

Answer 3

Mg2+

Question 4

What are the precursors added to make mrna

Answer 4

Ribonucleotide triphosphates

RATP rUTP rGTP rCTP

Question 5

What 2 consensus sequences are recognised by a holoenzyme at the promoter region

Answer 5

-35 and at -10

Negative because start site is +1

Question 6

Why are consensus sequences called hexameric

Answer 6

6 bases each

Question 7

What does strength of a promoter depend on

Answer 7

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)

Question 8

What is rna polymerase made of (subunits)

Answer 8

2 x alpha
1 x beta
1 x beta’
1 x w (omega)

Added sigma factor to produce a holoenzyme

Question 9

Why is the production of a holoenzyme needed

Answer 9

Sigma is needed to direct recognition of promoter -35 and -10 and rna polymerase can bind

Question 10

What gene is sigma 70 produced by

Answer 10

rPON gene

Question 11

Why are there alternate sigma factors to sigma 70 and give example

Answer 11

To match the cellular environment

For example sigma H is for heat shock genes in response to heat shock of a bacterium

Question 12

What is the complex of the holoenzyme and dna called in INITIATION and what property does it have

Answer 12

Closed complex - unstable

Question 13

Open complex occurs in elongation. What happens

Answer 13

Sigma factor opens DNA forming a transcription bubble at the +1 site

Rna starts to incorporate rNTPS

Question 14

When is sigma factor released

Answer 14

In elongation when the transcription bubble moves along to form 5’ to 3’ mrna

Question 15

How does elongation occur

Answer 15

Catalysis of phosphodiester bonds growing 5’ to 3’ between rNTPS depending on bad pair with template strand

PPi (pyrophosphate is lost)

Question 16

What other technique is used instead of proofreading by exonucleases like dna pol to try and reduce error rate in base pairs

Answer 16

Backtracking - breaking previous base pairs (not favoured)

Question 17

What 2 types of terminations is there?

Answer 17

Rho factor independent

Rho factor dependant

Question 18

What bases makes up factor independant terminator/termination of transcription

Answer 18

AT base pairs following GC palindromic sequences

Question 19

What shape does mrna produce due to rho independent pallindromic sequence

Answer 19

It forms a stem loop shape (via G-C base pairs)

Mrna forms BP with itself

Question 20

How does folding of mrna into stem loop cause weak interaction with dna allowing mrna to detach?

Answer 20

Only A U base paired with dna which is week and causes mrna to detach

Question 21

Why does polymerase detach from dna in rho independent termination

Answer 21

It becomes unstable on the helixes when mrna detaches

Question 22

What is rho factor made up of and what is its job

Answer 22

6 helical subunits

It unwinds rna-dna or rna-rna interaction

Question 23

What is unwinding by rho factor dependant on

Answer 23

ATP hydrolysis

Question 24

What occurs in rho dependant termination

Answer 24

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

Question 25

What is RUT site ?

Answer 25

Rho utilisation site C rich region on mrna helping it bind

Question 26

What 2 ways is gene regulation response to change in cellular environments

Answer 26

1- need different genes expressed 2- different level of genes expressed needed

Question 27

What is the repression strategy

Answer 27

Repressor molecule has a negative effect on expression It stops rna polymerase binding at the -35 -10 promoter sequence = no transcription

Question 28

How does activation strategy work

Answer 28

A protein activator is needed to bind to rna polymerase to help it bind to dna

Question 29

Why do protein activators be needed for rna pol to bind

Answer 29

Because it is a weak promoter sequence = rna not able to bind efficiently

Question 30

What happens in terms of rna polymerase when promoter sequences are ‘weak’ - ie not close to consensus

Answer 30

They are not recognised easily

Question 31

What is the lac operon (example of gene regulation)

Answer 31

A cluster of genes which are under control of a single promoter

Question 32

Name the components of the lac operon including what enzymes they code for

Answer 32

Plac - promoter region Lac operator (start site +1) Lac z - b galactosidase Lac Y - permease Lac A- acetylase

Question 33

WhT do the cluster genes on lac operon help ecoli to do

Answer 33

Allow use of lactose for carbon source instead of glucose

Question 34

What gene produces the lac repressor which inhibits lac ZYA expression

Answer 34

PlacI gene

Question 35

Explain the structure of the lac repressor and the 3 domains it has

Answer 35

Homotetramer Dna binding domain Regulatory domain Homotetramisation domain

Question 36

What does the homotetramer lac repressor bind to which stops gene expression of ZYA? By stopping rna recognition

Answer 36

The lac operator (start site +1)

Question 37

What does the lac operator consist of in its sequence and does it bind with high affinity to lac repressor?

Answer 37

It’s a palindrome sequence It binds to lac depressor tightly

Question 38

When is the transcription of the lac operon genes initiated?

Answer 38

When lactose is present When glucose isn’t present (would be preferred c source)

Question 39

What binds to the lac repressor which lowers its affinity to the lac operator site?

Answer 39

Allolactose (only present in presence of lactose)

Question 40

Explain the way lactose going into cell produces allolactose which can then start gene expression

Answer 40

Lactose enters via galactose permease The b galactosidase then turns it into allolactose (Or it can be hydrolysed into galactose and glucose)

Question 41

Is transcription fully blocked by the lac repressor

Answer 41

No there is still a small amount of transcription of ZYA genes

Question 42

How does glucose at low levels influence gene expression

Answer 42

Glucose at low levels is not enough to inhibit adenylate cyclase More cAMP is produced

Question 43

How does cAMP presence in low glucose levels cause gene expression

Answer 43

Camp binds to catabolite activator protein This then binds to rna polymerase and helps it recognise and bind to the weak promoter sequence

Question 44

Name the lac activator protein bound to cAMP

Answer 44

Catabolite activator protein CAP

Question 45

Why is both the activator (low glucose) and allolactose (high lactose presence) needed for gene expression

Answer 45

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

Question 46

What is the difference between allolactose and lactose

Answer 46

The alpha glycosidic bond is at 1-6’ instead of 1-4

Question 47

How does allolactose change the shape of the lac repressor lacI

Answer 47

Seperates the dna binding domains = lower affinity for the lac operator 1