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YEAR 2 S1 MOLECULAR > Mayerhoff > Flashcards

Mayerhoff Flashcards

1
Q

RNA polymerase II does what

A

mRNA transcription

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2
Q

how does euk RNA Pol II work

A
  • DNA strand is separated (slightly bent to enable separation)
  • RNA synth in active centre
  • RNA transcript comes out of enzyme
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3
Q

what is the role of the clamp domain in RNA Pol II

A
  • when RNA Pol II engages with DNA, the clamp domain sits MORE TIGHTLY on the enzyme thus BINDING IT
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4
Q

what is the role of the clamp domain in RNA Pol II

A
  • when RNA Pol II engages with DNA, the clamp domain sits MORE TIGHTLY on the enzyme thus BINDING IT
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5
Q

what is the CARBOXY-TERMINAL DOMAIN (CTD) in RNA Pol ll

what is its role

A
  • a seq of SEVEN aa that is repeated 25 TIMES in yeast and 52 TIMES in vertebrates
  • PHOSPHORYLATION SITE (fo those aa) important for REGULATION of enzyme
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6
Q

to transcribe a gene, RNA Poll II has to do what

A 
INITIATION 
1) recognise a start point in dsDNA: PROMOTOR 
2) BIND promotor
3) SEPARATE DNA strands 
4) INITIATE transcription at start site 
ELONGATION 
1) elongate until stop signal
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7
Q

what is a promotor

A

a DNA seq that determines the site of transcription INITIATION for a RNA Pol

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8
Q

what are the 3 main types of promotors in euk

A

1) TATA box
2) Initiator
3) CpG islands

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9
Q

describe TATA boxes

A
  • strong
  • sharp
  • 10-15%
  • inducible genes, prevalent in highly transcribed genes
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10
Q

describe Initiator complexes

A
  • variable
  • sharp
  • 15-30%
  • inducible genes, usually lacking TATA box
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11
Q

describe CpG islands

A
  • weak
  • broad
  • 60-70%
  • genes transcribed at a low rate (eg housekeeping genes)
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12
Q

the TSS (transcription start site) is what

A

+1

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13
Q

where is the TATA box

A

~30 bases BEFORE the TSS

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14
Q

give examples of genes that TATA boxes could act as a promotor for
mutations in the TATA box cause what

A
  • highly transcribed genes O
  • eg viral genes, cell cycle components, tissue specific genes
  • mutations within the seq ABOLISHES function
  • downstream/upstream movement of the TATA box causes shift the TSS
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15
Q

Initiator is what in comparison with the TATA box

A

an ALTERNATIVE

MUTUALLY EXCLUSIVE

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16
Q

where is the Initiator promotor found

A
  • -2 to +4 (O encompasses TSS)
  • if Initiator is moved up or downstream, the TSS moves too
  • Initiator is less well defined
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17
Q

describe the precision of CpG islands

how common are they
whatare they rich in

A
  • less precise TSS
  • 70% of genes
  • housekeeping genes
  • GC rich (O less bendy)
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18
Q

what is req for transcription initiation (“Construction” of the polymerase complex on the promoter)

A
  • RNA Pol

- General Transcription Factors (GTFs)

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19
Q

what is the function of GTFs

A
  • position RNA Pol II on the promotor
  • help separate DNA strands for transcription
  • form the PREINITIATION COMPLEX: GTFs & RNA Pol II
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20
Q

what is the role of TFIID

A
  • a GTF, it is the FIRST protein to bind DNA in formation of pre-initiation comple
  • 14 different subunits (O big)
  • cont TBP (TATA Box Binding Protein)
  • TBP= interacts with minor groove of DNA, causing BEND of DNA
    it is the first protein to bind DNA in formation of pre-initiation comple
  • TAFs (TBP Associaed Factors): recruit to TATA-less promotors (the promotors that don’t use TATA box), contacts to Initiator and DPE
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21
Q

size

what is the role of TFIIA

A
  • 2 subunits
  • binds DNA UPSTREAM of TATA box AFTER TFIID has bound
  • binds to TBP
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22
Q

size

what is the role of TFIIB

A
  • monomer
  • binds DNA on EITHER side of the TATA box AFTER TFIIA
  • binds to TBP
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23
Q

TFIID, TBP, TFIIA ANDTFIIB form what together

A

upstream promotor complex

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24
Q

after the upstream promotor complex is formed, what happens

what is formed

A

RNA Pol II joins

  • it forms a Pol II/ TFIIF complex
  • this complex then binds to the UPSTREAM promotor complex
  • positions Pol II over the TSS
  • forms the CLOSED PREINIIATION COMPLEX (PIC)
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25
Q

after the CLOSED PREINITIATION COMPLEX is formed, what happens next

A
  • TFIIE (heterodimer) binds to Closed Preinitiation complex

- creates docking site for TFIIH

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26
Q

TFIIF is what size

A

heterodimer

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27
Q

after TFIIE has bound, what happens

A

TFIIH binds

  • 10 subunits
  • cont HELICASE (unwinds DNA)
  • cont KINASE (phosphorylate RNA Pol II…?)
28
Q

why is it called a closed preinitiation complex

A

because DNA is still mostly in a DOUBLE HELIX

29
Q

at every promotor how many peptides are present

A

33 peptides

combined mass of 1.5MDa

30
Q

what is the order of TFII binding

A

D A B F E H

31
Q

how do we transition from transition to transcription

A

by going from CLOSED PREINITIATION COMPLEX to OPEN PREINITIATION COMPLEX (PIC)

32
Q

what happens to form the Open Preinitiation complex

A
  • TFIIH HELICASE subunit unwinds DNA (req ATP)

- this pushes back on the promotor bound GTFs, forming transcription bubble

33
Q

how is the INITIALLY TRANSCRIBING COMPLEX formed

A
  • TFIIB releases RNA exit channel
  • TFIIH KINASE activity phosphorylates CARBOXY-TERMINAL DOMAIN (CDT) of Pol II
  • recuits capping factors
  • TBP/TFIID are left at promotor
  • other GTFs dissociate
34
Q

the DNA template is read from which direction

A

3’->5’

35
Q

for PAUSING of transcription which factors are req

A

1) NELF (negative elongation factor)

2) DSIF (DRB Sensitivity Inducing Factor)

36
Q

which factor makes transcription continue

A

P-TEFb= cyclin-CDK

  • phosphorylates NELF and DSIF
  • phosphorylates Poll II CTD
  • causes Pol II to switch to ELONGATION mode, enzymes become processive, splicing factors also recruited
37
Q

give an example of a viral example of the use of RNA Pol II

what is the significance for treatment

A

HIV uses RNA Pol ll

  • maximises elongation efficiency
  • TAR hairpin formed (RNA base pairs with itself)
  • binds Tat protein
  • recruits P-TEFb (positive factor to help overcome neg factors)
  • switches to elongation

DRUGGABLE TARGET?

38
Q

what are proximal promotor elements

A
  • seq close to TSS, position may affect activity
  • short ~6-10bp
  • often direction INDEPENDENT
  • often TISSUE SPECIFIC
39
Q

what are distal “enhancer” elements

A
  • far from promotor(up to 50,000bp up/downstream or intronic)
  • typically ~50-200bp, but composed of several functional elements
  • direction independent
  • usuallu tissue specific
40
Q

whta is a transcription factor

A

transcription factor: protein (other than RNA Pol) that initiates or regulates transcription in eukaryotic cells

41
Q

specific transcription factors are what

A
  • Transcription Activators or Transcription Repressors
  • Stimulate or inhibit transcription of particular genes by binding to their regulatory sequences
  • Many: ~1,400 in human genome
  • Modular domain structure
42
Q

GTFs are for what

A

combining with RNA Pol ll to make the preinitiation complex

43
Q

what is the structure of a specific transcription factor

how are domains linked

A

2 main domains

  • DNA binding domain: recognises specific seq
  • an ACTIVATION or REPRESSION domain: alters transcription rates

linked by: flexible, disorded protein domain

44
Q

describe DNA binding domains in specific transcription factors
give an example of a DNA binding domain, where is it found

A
  • often DIMERS: homo/hetero
  • NON-COVALENT INTERACTIONS are SEQ SPECIFIC: H bonds, ionic interactions
  • a helix inserts into MAJOR GROOVE of DNA

eg helix-turn-helix in Bacteriophage 434 repressor

45
Q

describe Zinc-finger proteins

what are they

A
  • a DNA BINDING DOMAIN
  • short seq folds around co-ordinated Zn2+
  • cysteine and histidine side chains
46
Q

what are the 2 types of Zinc-finger proteins

A

C2H2 zinc finger

  • most common on humans
  • monomer

C4

  • in ~50 transcription factors
  • steroid receptor superfamily or nuclear rceptors
  • homo/heterodimer
47
Q

whata re Leucine-zipper proteins

describe them

A

DNA BINDING DOMAIN

  • extended alpha helices
  • basic residues interact with DNA backbone in major groove
  • coiled coil dimerization (stabilises by HYDROPHOBIC interactions between monomers)
  • can form heterodimers
  • dimers ‘grip’ DNA
48
Q

what are basic helix-loop-helix (bHLH) proteins

describe them

A

DNA BINDING DOMAIN

  • bind at N-termini
  • non-helical loops from leucine zipper-like coiled coil dimerization domains
  • can form heterodimers
49
Q

describe DNA binding activation domains

A
  • structurally diverse
  • often rich in one type of aa
  • may have intrinsically disordered conformation, but become more ordered with a co-activator
50
Q

what do many transcription factors bind as?

why are transcription factor interactions important

A

DIMERS

- transcription factor interaction increasse gene control options

51
Q

what effect does co-operation of monomeric transcription factors have

A
  • inc affinity

- stabilises binding to composite site

52
Q

what is the enhancosome

A

Multiple transcription factors co-operate at distant regulatory sites

53
Q

what regulates regulators

A
  • the transcription of TRANSCRIPTION FACTORS themselves is regulated
  • LIGAND BINDING: interaction w/ ligands eg drugs
54
Q

explain the role of nuclear receptors and ligand binding in

A
  • interact with steroid hormones
  • have a DNA binding domain and activation domain and ligand binding domain which make TF
  • bind w/ LIPID SOL HORMONES
  • family of ~50 transcrtiption factors
  • bind as HOMO or HETEROdimers
    C4 zinc finger proteins
55
Q

what does ligand binding do to nuclear receptors

A

changes subcellular localisation

because TFs can only bind DNA if they are in the NUCLEUS

56
Q

how is subcellular localisation of TFs changed

A

in cytosol (O cannot bind DNA)

  • before hormone binds to TF, the TF interacts with CHAPERONES, which prevent TF from moving to nucleus
  • hormone diffuses into cell, and binds to LIGAND BINDING DOMAIN of TF
  • causes CHANGE IN SHAPE, conformational change of LF domain
  • O free from chaperones
  • O can move to nucleus
  • DNA binding domain can now bind
  • Activation domain can activate gene
57
Q

what are the 3 basic mechanisms that underoin transcriptional activation

A

1) Modulation of chromatin structure
- impacts the ability of general transcription factors to bind
2) Recruitment of RNA polymerase
- Via mediator complex or recruitment of general transcription factors (TFIID)
3) Stimulation of elongation
- Via recruitment of P-TEFb or other elongation factors

58
Q

how is chromatin structure modulated

A

more tightly packed means less access for TF and RNA Pol II

59
Q

heterochromatin describe

A
  • inactive genes
  • more condensed
  • dark staining
  • histone deacylation
  • centromeres, telomeres
60
Q

histones being more acetylated means what

A
  • LESS densely packed

- O more accessible

61
Q

histone acetlyl transferases make DNA more/less accessible

A

MORE

62
Q

which TF is used to repress transcription

A 
HISTONE DEACETYLASE CO-REPRESSOR (HDAC)
- recruited to promotor via binding to transcription REPRESSOR DOMAIN 
- causes HYPOACETYATION 
- inc DNA-HISTONE interaction 
- inc packaging to 30nm fibre 
thus preventing GTFs binding
63
Q

which TF is used to activate transcription

A 
HISTONE ACETYL TRANSFERASE (HAT)
- co-activator receruited to promotor via binding to TRANSCRIPTION FACTOR ACTIVATOR DOMAIN 
- causes HYPERACETYLATION 
- dec DNA-HISTONE interaction 
- dec packaging to 30nm fibre 
thus allowing GTFs to bind
64
Q

if DNA is too tightly packed for even TFs to reach, how is it accessed

A

PIONEER TFs

  • bind one side of DNA helix even if DNA is wrapped around histone octamer
  • recruit histone acetlyl transferase
  • recruits more TFs once DNA= partially accessible
65
Q

how is RNA Pol ll recruited

A
  • via activation domains that recruit TFII
  • use MEDIATOR (connects RNA Poll ll and TFs)
  • mediator of transcription
  • molecular BRIDGE to RNA POL II
  • multiple subunits : some essential to all genes, some specific to 3-10% of genes
66
Q

how is the mediator recruited

A
  • activation domain interacts with MEDIATOR
  • mediator can simulatneously contact multiple activators (cooperativity)
  • DNA looping: enhancers
67
Q

incorrect transcription or transcription of mRNA that should not have been transcribed can cause what

A
  • multiple toes (dominant HOXD13 mutation)

- multiple wings on fly

YEAR 2 S1 MOLECULAR (7 decks)

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