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PM 228 - Gene expression > Lecture 1 > Flashcards

Lecture 1 Flashcards

1
Q

What is a haploid?

A

One genomic copy of each gene is present in the cell

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2
Q

What is the main control point for gene expression?

A

Transcription

_________
There are some limited opportunities for control of gene expression at translation

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3
Q

In which organism is transcription is immediately followed by translation?

A

Prokaryotes

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4
Q

in which organism transcript and translation are separated from each other in space and time?

A

Eukaryotes

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5
Q

How many kinds of RNA polymerase enzyme do bacterias have?

A

1 which transcribes ALL types of RNA

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6
Q

What are the sub-units of RNA polymerase?

A

alpha:

  • Function, assembly
  • Gene, rpoA
  • Number, 2
  • Mass(kd), 37

beta:

  • Function, catalysis
  • Gene, rpoB
  • Number, 1
  • Mass(kd), 151

beta prime:

  • Function, binds DNA
  • Gene, rpoC
  • Number, 1
  • Mass(kd), 155

sigma70:

  • Function, initiation
  • Gene, rpoD
  • Number, 1
  • Mass(kd), 70
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7
Q

What is an apoenzyme?

A

Multisubunit assembly: alpha(2),Beta,Betaprime

It can elongate and RNA strand but it CANNOT initiate transcription

For initation of transcription, promoter site in the DNA template must be recognised by the sigma subunit in the holoenzyme, alpha(2),Beta,Betaprime,sigma

Apoenzyme will combine with sigma subunit to form a holoenzyme.

After initiation, sigma factor will dissociate
-proved by 60bp -> decreased to 30bp footprint when sigma departs after first phosphodiester bond of RNA is formed

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8
Q

How are specific binding sites for a protein on double stranded DNA identified?

A

1) Gel retardation assay
2) Nuclease-protection assay
3) Footprinting assay

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9
Q

What is Gel retardation assay?

A

Electrophoresis of DNA fragments and migration through an agarose gel.

Samples can be labelled with P

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10
Q

What is nuclease protection assay?

A

DNA flanking the specific binding protein is completely digested (not protected)

RNA polymerase is used to protect the binding site of DNA, nuclease is used to cleave non-protected sites of the DNA.

The protected DNA is then released for analysis

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11
Q

What is footprinting assay?

A

Digestion by DNAse I

DNA is end-labelled by 32P

2 samples are prepared:

  • contains RNA polymerase
  • not contain RNA polymerase

DNAse is used to create nicks in the DNA, effectively creating fragments

Each sample is electrophorised in different wells, looking for missing protein fragments.

The missing protein fragments are binding sites for RNA polymerase

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12
Q

What is “The double footprint”?

A

A unique feature of RNA polymerase (holoenzyme).
It binds the site ~10 and ~35 regions of the promoter.

~35 region consensus:
TTGACAT

~10 region consensus:
TATAAT

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13
Q

What determines the specificity of the double footprint?

A

The sigma70 subunit.

The sigma70 subunit has important conserved regions

Thr/Gln binds to T
Trp binds to T
Tyr binds to T

The N-terminus controls the DNA binding

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14
Q

What is the Transcription “bubble”?

A

The unwound area of DNA where transcription occurs

~17bp opened (1.6 turns of double helix)

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15
Q

Which way does RNA polymerase work?

A

Chain elongation in direction 5’ -> 3’ with elimination of pyrophosphate from each incoming nucleotide

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16
Q

What is the DNA coding (sense) strand?

A

reads the SAME as mRNA (T>U), used for protein coding

17
Q

What is the DNA template (Anti-sense) strand?

A

Acts as a template for synthesis of mRNA, so this complementary in sequence to mRNA and also to Coding (sense) strand of DNA

18
Q

What is the significance of Asymetric enzyme?

A

Fit to asymetric promoter sequence.
Only in one orientation.
This defines which strand of double stranded DNA will be the template and hence direction of transcription.

19
Q

What is the first nucleotide at 5’ end of RNA?

A

pppA or pppG

20
Q

What is the difference between DNA polymerase and RNA polymerase?

A

RNA polymerase does NOT require primer or proof-reading

21
Q

What are the reactions involving RNA polymerase?

A

Similar to DNA polymerase

1) Nucleophilic attack by 3’-OH at the end nascent RNA strand, on alpha phosphate (5’-P) of incoming nucleotide.
2) Elimination and hydrolysis of PPi

22
Q

What is the function of sigma70?

And their consensus sequence?

A
  • Gene, rpoD
  • Use, housekeeping,consensus

~35 sequence:
TTGACA

~10 sequence:
TATAAT

16-18bp separation

23
Q

What is the function of sigmaS/38:

A
  • Gene, rpoS

- Use, Stress,Stationary phase

24
Q

What is the function of sigma32:

And their consensus sequence?

A
  • Gene, rpoH
  • Use, heat shock, chaperones

~35 sequence:
CCCTTGAA

~10 sequence:
CCCGATNT

13-15bp separation

25
Q

What is the function of sigmaE

A
  • Gene, rpoE

- Use, heat shock, extreme

26
Q

What is the function of sigma54

And their consensus sequence?

A
  • Gene, rpoN
  • Use, nitrogen starvation

~35 sequence:

~10 sequence:

27
Q

What is the function of sigma28/F

And their consensus sequence?

A
  • Gene, fliA
  • Use, Flagellar synthesis

~35 sequence:
CTAAA

~10 sequence:
GCCGATAA

15bp separation

28
Q

What is the housekeeping/vegatative growth sigma factor of Bacillus subtilis?

A

sigma43/A

29
Q

Outline out sporulation occurs

A

1) DNA replication
2) Septum formation
3) DNA translocates into forespore
4) Spore is engulfed
5) Spore coat forms
6) Mother cell is lysed
7) Spore released

30
Q

What sigma factors are used in forespore and mother cells?

A

Forespore: 43 > F > G
Mother: 43 > E > K

31
Q

How does reaction catalyzed RNA polymerase occur?

A

Similar to DNA polymerase, nucleophilic attack, by 3’ - OH end of nascent RNA stand, on alpha phospahte (5’-P), followed by elimination and hydrolysis of Ppi

32
Q

What is Coordinated control?

A

Control of related sets of genes by different digma factors specific for their promoters

33
Q

What is temporal control?

A

Control of successive stages of a complex process by hierarchies (or cascades) of sigma factors.

PM 228 - Gene expression (8 decks)

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