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Biotechnology > Control of Gene Expression > Flashcards

Control of Gene Expression Flashcards

1
Q

What are the types of RNA polymerase found in prokaryotes and how are they different?

A

Core

Holo - contains sigma subunit

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2
Q

What are the core components of prokaryotic RNA polymerase?

A

Beta subunit
Beta prime subunit
Alpha subunits (2)

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3
Q

Which subunit of prokaryotic RNA polymerase binds to the promoter region?

A

Sigma

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4
Q

Which part of the prokaryotic RNA polymerase subunit helps to stabilise RNA polymerase on the RNA?

A

The alpha subunits

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5
Q

What are the three different ways that RNA polymerase can be stabilised on the RNA?

A

alpha subunits

  1. UP element binding
  2. CAP (catabolite activating protein) class 1
  3. CAP class 2
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6
Q

What is the UP element and how does it work?

A

UP element is located before -35 region (on 5’ end - before start of translation). It binds the the C-Terminal Domain (CTD) of the alpha subunits of RNA pol. to stabilise it on the RNA.

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7
Q

What is CAP and how do the different classes work?

A

For CAP - DNA binding domain at C-terminus
Stabilises the pol. on the promoter

  • 60 is first CAP binding site (22bp of DNA) IN LAC Z
  • 40 is first CAP binding site, GalR(repressor) at -60 IN galP1

CLASS 1 = only CTD
CLASS 2 = CTD and NTD of RNA pol.

CAP has two regions conforming to transactivation domain, one binds to alpha c-terminal domain of RNA pol and other binds to n-terminal domain of RNA po

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8
Q

What happens if different activating regions (AR) of CAP are altered?

A

AR1 - does affect formation of closed complex, reduces recruitment (RNA pol. binding), but does not affect closed>open complex rate

AR2 - affects opening up of RNA for transcription (isomerisation rate) - closed>open complex (melting of DNA), formation of closed complex not affected (DNA-RNA pol. binding)

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9
Q

What are AR1 and AR2 and what do they do?

A 
AR = activation region
in class 2 CAP binding , RNA pol. binds to AR1 and AR2.

AR1 = forming closed complex between RNA and RNA pol. (stabilising)

AR2 = opening up RNA pol. from closed complex for transcription

PROKARYOTES

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10
Q

Components (basic) of a prokaryotic gene

A 
CAP site/UP site
Promoter 
Operator 
Structural Gene
Transcription Factors
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11
Q

What is an operon?

A

Genes of similar function ordered into groups, controlled by single promoter.

WHY?: Because they are likely in response to a particular condition where they would all be needed

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12
Q

Types of Histone Modification

A

Phosphorylation
Acetylation - neutralises charge (tails are +ve charge/acetyl groups -ve charge)
Methylation
Ubiquitination

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13
Q

Promoter region in Eukaryotes

A

-30 TATA box

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14
Q

Promoter Region in Prokaryotes

A

-35 and -10 (TATA equivalent) sites

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15
Q

What is the intermediate region between the promoter regions in prokaryotes?

A

Important - 15-21 BP long
May alter how RNA pol. binds to RNA
WHY? 10 BP per turn of alpha helix, length of region -> whether both promoter regions are on the same side for RNA pol. binding

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16
Q

How does bacteria have differential gene expression?

A

Change of sigma factor decides promoter that RNA pol. binds to.

How? Slightly different sequence for promoter binding area.

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17
Q

IMPORTANCE OF SIGMA FACTOR (EVIDENCE):

A
  1. CROSS-LINKING
    MIX RNA + PROMOTER SEQ. +UV LIGHT
    SEE WHERE STICKS
    CROSS-LINKS BETWEEN SIGMA AND DNA
  2. SUPPRESSOR OR ‘SECOND-SITE’ MUTATIONS
    MUTATE TO AFFECT THE ABILITY OF RNA POL. TO BIND TO PROMOTER
    SELECTING FOR RNA POL. THAT CAN RECOGNISE PROMOTER
    FIND OUT WHERE SUPPRESSOR MUTATIONS ARE (WHERE DOES NOT WORK)
    SIGMA FACTOR
  3. DELETING THE N-TERMINUS OF SIGMA FACTOR ALLOWS DNA BINDING
    PART OF SIGMA FACTOR INHIBITS BINDING (N-TERMINUS)
    REMOVE N-TERMINUS -> EASIER BINDING
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18
Q

TBP

A

Tata binding protein (TO TATA BOX)

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19
Q

How is TBP unusual?

A

Does not bind using alpha helices

Binds using beta-sheet secondary structure in minor groove of DNA (in other cases - with alpha would by major groove)

-> Distortion, bending and binding of DNA (-> melting)

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20
Q

THE STEPS - PRE-INITIATION COMPLEX

Eukaryotes

A
  1. TBP binds
  2. TB2F binds
  3. TF2E binds
  4. TF2H binds

Eurkaryotes

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21
Q

TF2H role in the initiation complex

A

TFIIH (2H) - kinase CDK7 is a subunit, which phosphorylates C-terminal domain of RNA pol. II at serine 5 in transition from initiation complex to elongation complex

Eukaryotes

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22
Q

ELONGATION COMPLEX FORMATION

Eukaryotes

A

CTD very IMPORTANT

Synthesis mRNA - transcription factor dis-assemble, so RNA pol. Can transcribe
Phosphorylate large RNA pol. II subunit (RPB1 - large subunit)

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23
Q

What does CDK stand for in CDK9?

A

Cyclin-dependent kinase

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24
Q

How are CDKs involved in the Elongation Complex formation?

A

CDK9 & CDK12

Phosphorylate at serine position 2 of C-terminal domain (CTD of the RNA pol. II large subunit)

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25
Q

TERMINATION OF TRANSCRIPTION

Eukaryotes

A

FCP1 (bound with the RNA pol. II), phosphatase (de-phosphorylates), removes phosphate groups so RNA pol. II can recycle back to interact with general transcription factors FOR PRE-INITIATION

26
Q

What does RNA pol. 1 use for pre-initiation/elongation/termination steps?

A

SL1 (TBP)
UBF
Rm3p

27
Q

What does RNA pol. 3 use for pre-initiation/elongation/termination steps?

A

TFIIIC

TFIIIB (TBP)

28
Q

How does a repressor work?

Prokaryotes

A

DNA binding domain at N-terminus

  1. Bind to the promoter/close to promoter - PREVENT RNA POL. BINDING
  2. Binds in multiple places within operon and upstream from promoter causing looping - PREVENT RNA POL. BINDING
  3. Upstream of promoter and interferes with activation BLOCKS ACTIVATION INITIATION
29
Q

Where is the operator sequence on the gene?

Prokaryotic

A

Between the promoter region and the start codon.

30
Q

How does an activator work?

A

2 domains
Binds to DNA/ Activates transcription
At CTD or NTD of factor
Does something to DNA to make the polymerase work better

  1. binds upstream and stabilise RNA pol. on DNA
  2. binds closer to start site, regulate ability of RNA pol. to initiate
31
Q

How to find transcription factors

A 
DNA footprinting (e.g. DNase 1)
Chromatin immunoprecipitation
32
Q

How does DNA footprinting work?

A

E.g. will randomly chew up DNA
If nothing bound - random
If something bound, will protect that strand of DNA consistently
(use formalin to fix the proteins on to the DNA)

33
Q

How does chromatin immunoprecipitation work?

A

Cross-link the DNA (irreversible - permanent)

Fragment DNA

  • Sonication (random)
  • Restriction enzymes - selective

Add antibody specific to that transcription factor - pull down all the DNA seq.

Reverse cross-link

Get rid of protein (transcription factor), isolate DNA

Analyse DNA left

  • Quantitative PCR - that gene
  • Sequence - whole genome area of genome where that protein binds
34
Q

What is an enhancer and how does it work?

Prokaryotic

A
  1. Upstream and stabilise RNA polymerase on promoter (closed complex)
  2. Bind closer to start site on to OPERATOR SEQUENCE, regulated RNA polymerase ability to initiate

DOING SOMETHING TO DNA TO MAKE POLYMERASE WORK BETTER

35
Q

Control gene expression in the Lac operon

A

Can exist in three diff. states:

  1. Fully repressed - LAC REPRESSOR (BINDS TO OPERATOR SEQ.)
  2. De-repressed operon - STOPS REPRESSOR -> BASAL LEVEL
  3. Activator (cAMP) - binding of activator protein - HELPS RNA POL. BIND
36
Q

How can sugar concentrations control expression in the Lac operon?

A

FULLY REPRESSED -ve lactose/+ve glucose = repressor can bind, cAMP cannot

DE-REPRESSED +ve lactose/+ve glucose = repressor less effective, cAMP cannot bind (because glucose high)

ACTIVATED +ve lactose/low glucose = repressor less effective, cAMP can bind and activate transcription

37
Q

Example of an activator (prokaryotes)

38
Q

Example of a repressor

A

Lac repressor

39
Q

CONTROL OF GENE EXPRESSION - using sugar metabolism

A

Using Galactose in S. cerevisiae

REPRESSED - glucose present

Gal 2, 1, 7, 10 (break down galactose -> glucose (x2)) and so do not need to be transcribed AND associated transporter proteins 
Gal 4 (transcription factor) repressed by Gal 80

ACTIVATED - glucose not present so need the galactose metabolism
Require the structural genes, causes Gal 3 to be produced.

Gal 3 interacts with Gal 80 -> releases Gal4 from repression
> gene transcribing
(Gal4 interacts with TBP, TB2B and RNA pol.)

40
Q

What is Gal4?

A

a transcriptional activator that binds to UAS enhancer sequences found in DNA

recruits transcription machinery to the site to induce gene expression

eukaryotes?

41
Q

What is an enhancer?

Eurkaryotes

A

regulatory DNA sequences that, when bound by specific proteins called transcription factors, binding of an activator causes the transcription of an associated gene

42
Q

What is a terminator?

A

End of the transcription sequence (not stop codon), AU-rich, hairpin sequence

43
Q

What is polydeadenylation?

A

Adding a Poly(A) tail to mRNA in eurkaryotes

44
Q

What is the purpose of polydeadenylation?

A

Improving mRNA stability in eukaryotes

45
Q

What is Kovak’s sequence?

A

Conserved start sequence at the beginning of the open reading frame of mRNA in eukaryotic cells - for optimum transcription initiation.

46
Q

What is ubiquitination?

A

Type of histone modification
Usually done is response to DNA damage
Marks DNA for degradation

47
Q

What is acetylation?

A

Type of histone modification
Done to make DNA more available
Neutralises the charge (+ve) on histone tails, so weaker binding with DNA (slightly -ve)

48
Q

How do you identify the different types of RNA polymerase in eurkaryotic cells?

A

Alpha-amanitin inhibition
Low conc. (1mg/mL) RNA pol. II
Med conce. (10mg/mL) RNA pol. III
Resistance RNA pol. I

49
Q

What is the importance of the C-terminal domain of the large subunit of RNA polymerase II (eurkaryotes)?

A

Repeat of 7 amino acids
Serine 2 and serine 5 are the ones that are phosphorylated (elongation complex)

Number of repeats of this regions is unique to each species

50
Q

How did they test the importance of the C-terminal domain of the large subunit of RNA polymerase II (eurkaryotes)?

A

Removed the CTD and saw what happened, remove large and larger sections of it (truncation) - effect on transcription

51
Q

The important of RNA pol. crystal structures (between eukaryotes and prokaryotes)

A

Both have alpha helices

Similar 3D structure - groove for binding site

52
Q

What is meant by RNA pol. cannot recognise start of transcription?

A

Need structures to recognise

e. g. - transcription factors
e. g. - promoters

53
Q

What is special about mRNA translated from an operon?

A

Multiple genes in one mRNA strand, then it is expressed separately (internal ribosome binding sites)

Or cut post-translationally

54
Q

How to identify the sequence of a promoter region

A

Recognised and bound by RNA polymerase, so can fix and sequence what is fix-cut-release-sequence.

Can also seq. genes and compare the conserved regions near transcription start sites across genes -> consensus sequences

55
Q

How might you regulate a promoter

A

Length of intermediate region determines promoter strength for RNA pol.

Promoter regions on same side of helix = stronger binding structure

56
Q

When in DNA replication do repressors block transcription

A

Regulate closed complex (recruitment and binding of RNA polymerase)

Regulate close-open-initiation change

Regulated termination of transcription

57
Q

How does Gal4 enhance transcription?

A

Gal4 interacts with TBP, TB2B and RNA pol.

It assists in formation of closed complex.#

58
Q

Purpose of sigma factor

A

Binding to promoter region (lost when transcription starts)

59
Q

What is strength of promoter measured in?

A

Frequency at which RNA polymerase initiates transcription

- related to closeness of -10 and -35 regions & regulatory proteins & surrounding sequences

60
Q

Recruitment of chromatin modifying enzymes

A

Modifying packaging proteins (histones) transcription factor recruits different complexes of enzymes

Removal/addition of acetyl/methyl groups
Ubiquitination - marks DNA for degradation

Biotechnology (5 decks)

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