methods of transcription

Question 1

genome expression

Answer 1

50k genes
only 10k expressed in any given cell
genome identical in all cells
geneome must be differentially expressed

Question 2

differential gene expression regulation

Answer 2

time - devlopment and in response to hormones

space - different tissues express diff genes

Question 3

importance of differential gene expression ?

Answer 3

because gene expression controls metabolism / cell shape / motilitiy / differentiation and proliferation

failure to regulate would cause disease in these processes.

Question 4

differentiation from a single cell

Answer 4

totipotent cell to pluripotent to blood stem cells or other comitted cells

Question 5

reprogramming

Answer 5

comitted cells can be turned into pluripotent cells through reprogramming

Question 6

regulation of gene expression

Answer 6

transcriptional level - turning on/off dna to rna synthesis

post transcriptional - regulation of rna stability / translation

Question 7

which regulation level is more common ?

Answer 7

transcriptional is more common as it is more efficient to express 10k genes than express 50 and stop them from bein translated

Question 8

genes can be transcribed on different levels

Answer 8

abundant
rare
and no transcripts

Question 9

housekeeping and tissue specific genes ?

Answer 9

housekeeping - expressed in all cell types

tissue specific - expressed only in certain tissues.

Question 10

induicible genes

Answer 10

genes that normally dont transcribe anything but can be stimulated to transcribe abundantly

Question 11

eukaryotic rna polymerases

Answer 11

pol 1 pol 3 and pol 2

Question 12

what does pol 2 do

Answer 12

transcribes all protein encoding genes

Question 13

structure of pol 2

Answer 13

crystalised in yeast

dna enters complex through cleft and jaws will grip dna and open / close to allow dna movement dna hits wall which will cause conformational change and allows exit

Question 14

promoters

Answer 14

sequence immideately 5’ to transcription unit
promoters recruit rna polymerase to a dna template
rna polymerase only moves one way

Question 15

minimal rna pol 2 promoter

Answer 15

min bit of dna essential for transcription
2 types
core - containing tata - above start poin
tata-less core promoter - below start point

Question 16

consensus sequence

Answer 16

dna sequences that are found in multiple regions across different organisms,gebnes or regions e.g. TATA box

Question 17

key points of eukaryotic transcription

Answer 17

rna polymerase cannot recognise promotesr and need general transcritiption factors

GTFs + pol2 = basal transcription apparatus

to iniate transcription eukaryotic transcription complexes must gain access to DNA which is packed into nucleosomes

larges pol 2 subunit co ntains C-terminal domain - involved in regulation of transcription,initation,elongation and mRNA processing

Question 18

explain how pol 2 binds to tata box

Answer 18

pol2 forms basaltranscritipon appartus with TF2D
TF2D binds to TATA and recruites TF2B which provides binding surface for pol2 and is involved in start site determination
this recruites RNA polymerase 2 which has TF2F which is required for initiation and recruites TF2E and H.
TF2H binds and catalyses ATP - dependent unwinding of start stie DNA and phosphorylation of CTD.

Question 19

CTD - c terminal domain

Answer 19

largest subunit of pol2 - 52 repeats
when pol 2 is re4cruited it is hypophosphorylated and becomes hyperphosphorylated on serine 2 and 5 during transcription

Inhibitors of CTD phosphorylation prove its importance

Deletion of CTD in mice and yeast is deadly

phos of CTD required for promoter clearance and for recruitment of rna processing complex

phos by Specific Cyclin-dependent kinases

Question 20

ctd kinase activation

Answer 20

tf2h - bibnds to cdk 7 which pho serine 5 more than serine 2
others include c-m,yc and cdk9

Question 21

transcription factors

Answer 21

recruit rna polymerase to genes needed to be transcribed
bind specific dna sequence - causing transcription or prevent it

Question 22

regulatory elements

Answer 22

needed to regulate recruitment
found upstream tata box

Question 23

how is transcription regulated >

Answer 23

by efficiency of recruitment of rna polymerase 2

Question 24

steroid hormone signalling

Answer 24

steroid enters cell
goes to receptor protein
receptor - hormone complex enters nucleus
is a trancription factor
binds to specific dna and induces specific genes to hormone

e.g. oestrogen

Question 25

how to interfer with regulatory and general transcription factors

Answer 25

drugs - tamoxifen is an antagonist of oestrogen-responsive transcription

Question 26

transcriptional activatyors

Answer 26

attract pol 2 to promoter contain dna binding domain DBD and activation domain AD dna betwen promoter and enhancer loop out to enable the activation domain to contact promoter associated factors dna binding and activation domains are distince from one another and can be swapped

Question 27

what determines epcificity for activation

Answer 27

dna binding domain by beringing transcriptional activation domain to vicinity of promoter

Question 28

two hybrid screen

Answer 28

tells us if two proteins interact non interacting proteins tagged with dbd and AD x with dbd and y with ad if they dont interact rna pol 2 is not recruited and no expression if they interact rna pol 2 is recruited and expression occurs

Question 29

Activation domains

Answer 29

likely to mediate non specific p-p interactions glutamine rich proline-rich

Question 30

DNA binding domain

Answer 30

many common motif that mediate binding of transcription factors to dna like zinc finger helix loop helix coiled coil

Question 31

zinc finger motif

Answer 31

cycs2/his2 finger zing bound between 2 cycteine and 2 histidine fingers are 23 aa long usually found in tandem cys2 cys 2 fingwe non repetitive in proteins diff function per finger

Question 32

homeo domain

Answer 32

found in develop proteins 60aa long 3 helixes - bind tro major groove of dna

Question 33

how tf become active

Answer 33

protein synth ligan bind protein phos addition of second subunit unmasking- inhibitor unmasked stimulation of nuclear entry- allowed to enter nucleus release from membrane

Question 34

enhancers

Answer 34

dna sequences non immediately adjacent to where transcription starts enhance recruitment of rna polymerase to a promoter reside 5' or 3' to transcription unit very stong binding sites for transcription factors

Question 35

how is thwe dna made accessible

Answer 35

DRE binds to TF regulatory element is accessible direct recruitment or mediator or other things

Question 36

what is a mediator | a

Answer 36

a comples that transduces both negative and positive regulatory information from specific activators or repressors to core transcriptional machinery

Question 37

mediators

Answer 37

required to mediate response to activators does not bind to dna interacts pol2 via ctd has a kinasde module includes - cdk8 many forms most activate transcription some repress mediator + pol2 + GTFS = holoenzyme

Question 38

Silencers

Answer 38

dna sequences that inhibit rna polymerase can reside 5' or 3' of transcription unit

Question 39

transcription repressors

Answer 39

competitive DNA bind - binds to same binding site as activator masking the activation surface - 2 diff binding site for repressor and activator repressor make contact to activator and stops it recruiting pol2 direct interaction with GTF binds TF2D preventing it binding activator

Question 40

transcription coactivators

Answer 40

two type - closely associated with basal transcription machinery 2 modify chromatin structure - HATS and Chromatin remodelling complexes