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Genes to Proteins > Transcription & Control of Gene Expression > Flashcards

Transcription & Control of Gene Expression Flashcards

1
Q

What are the 3 types of bacterial RNA?

A

ribosomal (rRNA)
transfer (tRNA)
messenger (mRNA)

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2
Q

How do you determine long it takes for bacteria to divide with a doubling time of 20 min with 5x10^8 cells overnight?

A

2^n = 5x10^8 (n=intervals)
log2^n = 2log n = 5 x 10 ^8
n=17.7/0.693
=26 -> 13 hours

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3
Q

What does RNA polymerase need to work?

A

RNA synthesis needsa DNA template to work

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4
Q

How can we measure RNA pol activity?

A

Incorporate radio labelled rNTP into acid preciptable RNA

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5
Q

How fast and what direction does RNApol work?

A

transcribes 40-60 bases per second in the 5’ to 3’ direction

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6
Q

what are the phases of RNA transcription

A

initiation, elongation and termination

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7
Q

What are the steps of RNApol elongation?

A

1) Enters DNA downstream
2) DNA is unwound
3) mRNA transcribed to be complementary to the 3’ to 5’ DNA strand
4) mRNA leaves
5) RNA continues to go upstream

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8
Q

Describe the mechanism of the RNA polymerase catalysed reaction.

A

In the 5’ to 3’ direction, OH group in the nucleotide attacks the alpha Phosphorus, kicking of the pyrophosphate leaving group, forming a phosphodiester bond.

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9
Q

What is the role of sigma as a transcription initiation factor?

A

sigma factor helps RNA polymerase find promoter sequences and initiate transcription

once transcription is initiated, sigma dissociated and core enzyme continues into elongation of the transcript

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10
Q

How can antibiotics inhibit RNA polymerase?

A

Rifamycin - blocks initiation of trancription

Streptolydigin - blocks elongation of transcription

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11
Q

Now con you isolate antibiotic resistant mutants?

A

plant large numbers (10^8) of cells and use less than
optimal antibiotic concentrations

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12
Q

What specific changes happen to E. coli mutants to make them antibiotic resistant?

A

E. coli mutants were found to have identical α, β’, σ and ω RNApolymerase subunits but altered β-subunits, there is a single amino acid change, indicating β having a role initiation and elongation

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13
Q

What does each component of RNApol name in transcription?

A

β and β’ - form the catalytic centre

α and ω - required for assembly of the enzyme

α - aspects of promoter assembly

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14
Q

What is the process of defining transcription start sites?

A

A phosphate at the 5’ end of DNA is radiolabelled:

1) in vitro transcription with RNApol and rNTPs to make multiple transcripts of mRNA from the DNA template

2) denature heating

3) hybridise DNA and mRNA

4) S1 nuclease introduced, cleaving single stranded DNA (trims extra)

5) NaOH introduced to degrade mRNA as RNA is sensitive to base

6) determine DNA length to infer start site

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15
Q

Where and what are the bacterial promoters?

A

-10, pribnow box, TATAAT, essential for initiation
-35, TTGACA, controls rate of transcription

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16
Q

Describe the process of DNase Footprinting

A

A control is made where DNase cleaves the phosphorilated DNA at various positions, creating multiple fragments, observed via denaturing gel electrophoresis and audioradiography.

The same is run but with RNApol, and the ~70 base footprint region can be seen by comparing the missing fragments to the control, as RNApol binding obscured DNase cleavage

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17
Q

How can the lack of -35 region of promoter effect mRNA synthesis?

A

when observed with exonuclease, the DNA is degraded from the ends and -35 removed, a short mRNA transcript is produced but RNApol cant rebind as -35 required for binding but not initiation.

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18
Q

Describe the stem-hairpin loop structure

A

a palindromic sequence that runs in opposite directions and when folded into a hairpin loop, one side of the stem is complimentary to the other

it is a rho-independent terminator

has a G:C rich stem

is followed by a run of U’s (UUUUUU)

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19
Q

describe Rho-idependent termination

A

1) hairpin loop in mRNA causes RNApol to pause

2) weak dA-rU DNA-mRNA base pairing causes thermodynamic dissociation

3) RNA dissociates from DNA

4) mRNA dissociates from RNApol

5) DNA strands recombine as transcription bubble closes

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20
Q

When is Rho-dependent termination needed?

A

When mRNA is not followed be a run if U’s

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21
Q

What is Rho and Describe Rho-dependent termination

A

Rho is a protein of 46 kDa and is hexameric

It binds to RNA at rut (rho utilisation) sequences and tracks along mRNA in the 5’ to 3’ direction

Its helicase activity separates mRNA from the DNA template

22
Q

Define constitutive gene expression

A

Genes whose levels of transcription are determined solely by the strength of the interaction of RNA polymerase with the promoter (K) are said to be constitutively expressed

23
Q

How do E. coli utilise lactose

A

When lactose is introduced to the cell, lacZ (beta galactosidase) rises 1000 fold.

lactose can then be hydrolysed into galactose and glucose or isomerised into allolactose

24
Q

Describe the lac operon

A

First there is the promoter followed by short (38 kDa) transcribed lacI (lac repressor) which is then terminated

Then there is a second promoter and then operator which the transcribes 3 proteins of lac mRNA:

lacZ - beta-galactosidase (125 kDa)
lacY - lactose permease (30 kDa)
lacA - beta-galactoside transacetylase (30 kDA)

25
Q

What is the half life (t 1/2) of lac mRNA?

A

3 minutes so the system can respond quickly to appearance or depletion of lactose

26
Q

Is lactose a beta-galactosidase substrate or inducer?

A

Substrate

27
Q

Is isopropyl-beta-D-thiogalactose (IPTG) a beta-galactosidase substrate or inducer?

A

inducer

28
Q

Describe the DNase footprinting of LacI (lac repressor)

A

all DNA marked at 5’ end with radiolabelled phosphate

i) control of lacPODNA and DNase, breaks into fragments which are observed with denaturing gel electrophoresis

ii) lacPODNA + DNase + LacI (lac repressor)

iii) lacPODNA + DNase + lacI + IPTG

isopropyl - beta - D - thiogalactose (IPTG) prevents cleavage

when all fragments compared, the missing section in ii shows the footprint of lacI (lac repressor)

29
Q

What is the lac control region?

A

The overlap of RNA polymerase and lacI (lac repressor)

in this space, genes are switched off until the introduction if lactose

30
Q

How can you identify the lac operator?

A

there is a palindromic sequence called the “synthetic olgionucleotide duplex”

31
Q

How does the lac operator structure help its function?

A

it has looping which allows lac repressor to bind in multiple places

32
Q

How do the concentrations of glucose and cAMP in an E. coli cell relate to each other?

A

they are inversely correlated (when one goes up the other goes down)

33
Q

Where is the activator site in the lac control region?

A

Upstream of RNA polymerase as it promotes the process

34
Q

What 2 things are required for lac operon expression?

A

1) a positive stimulus by Crp

2) the absence if a negative stimulus from lacI (lac repressor)

35
Q

What is the role if Crp?

A

it binds to cAMP, this complex then can bind to the activator site

it also binds to other DNA sections to prevent any sugar other than glucose

36
Q

How do the sugars communicate to RNA polymerase?

A

through receptor proteins

37
Q

How can alternate sigma factors be useful?

A

they can recombine with core RNA polymerase and direct to distinct sets of promoters that they have the ability to recognise,, this then allows a new set of genes to be transcribed.

this is good for when large sets of genes that are only needed in particular physiological circumstances

38
Q

What are the 2 alternate lifestyles of bacteriophage λ?

A

lysis and lysogeny

39
Q

What does lysis require?

A

replication of phage particles i.e. viral proteins and DNA and rupturing of the host cell membrane

40
Q

What does lysogeny require?

A

requires the prevention of replication of phage particles, instead needs integration of the phage DNA into the bacterial chromosomal DNA

41
Q

What is the cos site?

A

“Cohesive” - has single strands on each end

42
Q

what is cI?

A

λ repressor. acts as a repressor at P(L) and P(R), and an activator at P(RM)

-turns off early genes, turns on its own gene

43
Q

what is cII?

A

activator of transcription from P(RE) and P(l), there is no transcription from these promoter unless cII is present

-turns on cI recombination genes

44
Q

what is cIII?

A

acts as a protector if cII, enhancing its stability from degradation

45
Q

what is Cro?

A

a repressor at P(RM)

as Cro concentration rises, it binds to and represses transcription at P(L)

binds to P(R) to turn off its transcription

46
Q

what is N?

A

an antiterminator of transcripts initiated at P(L) and P(R)

  • turns on cII, replication, Q, cIII and recombination genes (early genes)
47
Q

how do rich vs starved conditions effect if lysis or lysogeny is followed?

A

in rich conditions -
- cells grow, proteases active -> follows lysis
- new hosts available -> cII is degraded
- no cI from P(RE)

in starved conditions -
- cells not growing so no new hosts -> follow lysogeny
- absence if proteases -> cII is stable and active
- cI made from P(RE)

48
Q

How do cI and Cro bind to O(R) sites?

A

Cro-
has 10 fold higher affinity for OR3 than OR2 and OR1 sites

cI-
has 10 fold higher affinity for OR1 than OR2 and OR3

they fill the sites oppositely so that each first turns the others gene OFF

49
Q

What is special about cI binding and Cro binding?

A

they are cooperative (looks like theyre hugging)

50
Q

what us the function of lexA?

A

represses genes required to repair DNA in the SOS response

Genes to Proteins (3 decks)

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