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Biochem - Year 1 Biomed > Gene Expression/transcription(e Coli) > Flashcards

Gene Expression/transcription(e Coli) Flashcards

1
Q

Why do we study gene expressiom regulation in E. coli

A

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

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2
Q

What strand is used to produce mrna

A

Anti sense strand (template)

Antisense means opposite to mrna

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3
Q

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

A

Mg2+

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4
Q

What are the precursors added to make mrna

A

Ribonucleotide triphosphates

RATP rUTP rGTP rCTP

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5
Q

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

A

-35 and at -10

Negative because start site is +1

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6
Q

Why are consensus sequences called hexameric

A

6 bases each

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7
Q

What does strength of a promoter depend on

A

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)

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8
Q

What is rna polymerase made of (subunits)

A

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

Added sigma factor to produce a holoenzyme

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9
Q

Why is the production of a holoenzyme needed

A

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

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10
Q

What gene is sigma 70 produced by

A

rPON gene

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11
Q

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

A

To match the cellular environment

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

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12
Q

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

A

Closed complex - unstable

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13
Q

Open complex occurs in elongation. What happens

A

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

Rna starts to incorporate rNTPS

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14
Q

When is sigma factor released

A

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

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15
Q

How does elongation occur

A

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

PPi (pyrophosphate is lost)

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16
Q

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

A

Backtracking - breaking previous base pairs (not favoured)

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17
Q

What 2 types of terminations is there?

A

Rho factor independent

Rho factor dependant

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18
Q

What bases makes up factor independant terminator/termination of transcription

A

AT base pairs following GC palindromic sequences

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19
Q

What shape does mrna produce due to rho independent pallindromic sequence

A

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

Mrna forms BP with itself

20
Q

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

A

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

21
Q

Why does polymerase detach from dna in rho independent termination

A

It becomes unstable on the helixes when mrna detaches

22
Q

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

A

6 helical subunits

It unwinds rna-dna or rna-rna interaction

23
Q

What is unwinding by rho factor dependant on

A

ATP hydrolysis

24
Q

What occurs in rho dependant termination

A

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

25
Q

What is RUT site ?

A

Rho utilisation site

C rich region on mrna helping it bind

26
Q

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

A

1- need different genes expressed

2- different level of genes expressed needed

27
Q

What is the repression strategy

A

Repressor molecule has a negative effect on expression

It stops rna polymerase binding at the -35 -10 promoter sequence

= no transcription

28
Q

How does activation strategy work

A

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

29
Q

Why do protein activators be needed for rna pol to bind

A

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

30
Q

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

A

They are not recognised easily

31
Q

What is the lac operon (example of gene regulation)

A

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

32
Q

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

A

Plac - promoter region

Lac operator (start site +1)

Lac z - b galactosidase

Lac Y - permease

Lac A- acetylase

33
Q

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

A

Allow use of lactose for carbon source instead of glucose

34
Q

What gene produces the lac repressor which inhibits lac ZYA expression

A

PlacI gene

35
Q

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

A

Homotetramer

Dna binding domain
Regulatory domain
Homotetramisation domain

36
Q

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

A

The lac operator (start site +1)

37
Q

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

A

It’s a palindrome sequence

It binds to lac depressor tightly

38
Q

When is the transcription of the lac operon genes initiated?

A

When lactose is present

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

39
Q

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

A

Allolactose (only present in presence of lactose)

40
Q

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

A

Lactose enters via galactose permease

The b galactosidase then turns it into allolactose

(Or it can be hydrolysed into galactose and glucose)

41
Q

Is transcription fully blocked by the lac repressor

A

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

42
Q

How does glucose at low levels influence gene expression

A

Glucose at low levels is not enough to inhibit adenylate cyclase

More cAMP is produced

43
Q

How does cAMP presence in low glucose levels cause gene expression

A

Camp binds to catabolite activator protein

This then binds to rna polymerase and helps it recognise and bind to the weak promoter sequence

44
Q

Name the lac activator protein bound to cAMP

A

Catabolite activator protein CAP

45
Q

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

A

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

46
Q

What is the difference between allolactose and lactose

A

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

47
Q

How does allolactose change the shape of the lac repressor lacI

A

Seperates the dna binding domains

= lower affinity for the lac operator 1

Biochem - Year 1 Biomed (21 decks)

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