RR6: Molecular Mechanisms Driving Transcriptional Initiation

Question 1

how can you validate whether a given protein that we suspect might interact with a DNA sequence that we’re interested in (a cis acting regulatory element)?

Answer 1

carry out EMSA
based on the idea that proteins interact with DNA in a very specific manner
recognise these sequences and bind to the substrate (DNA)
we can identify those complexes because DNA substrate can be labelled
any time it interacts with the protein, this changes its mobility through an electrophoretic field
if you radiolabel it, can be seen clearly

Question 2

how can that be further tested/identified after EMSA?

Answer 2

carry out chromatography column
fraction it
test it to identify the proteins
add an antibody against the protein to obtain a supershift (change mobility even more)
do Chip seq to figure out exactly the sequence that is the cis regulatory element

Question 3

what are some domains in transcriptional activators and some characteristics?

Answer 3

have DNA binding domains and transcriptional activation domains
but those domains are very unstructured, hard to categorize, we don’t know much about them

Question 4

how do those transcriptional activation domains actually affect transcription?

Answer 4

bind to enhancers or proximal promoter elements through that interaction with DNA binding domains and cis acting element

Question 5

how many subunits in the mediator complex?

Answer 5

31 subunits

Question 6

where was the mediator complex initially identified?

Answer 6

in yeast, but homologs were found in humans

Question 7

how many major domains in the mediator and their names?

Answer 7

3 major domains
middle, head and tail

Question 8

where are all the mediator subunits localized?

Answer 8

localised into one of each of these domains

Question 9

what is a special characteristic of the head and middle domains?

Answer 9

they are flexible with respect to one another, and can take on a conformation that allows them to interact along one interface with RNA polymerase II

Question 10

what are some special subunits?

Answer 10

some subunits present within the domains interact specifically with DNA binding transcriptional activators

Question 11

what happens if you mutate one subunit?

Answer 11

it will not affect the overall activity of the mediator nor overall transcription but it will disable that specific transcriptional activation associated with a given proximal promoter binding transcriptional activator or enhancer binding transcriptional activator

Question 12

what can the mediator do in a chromatin loop?

Answer 12

acts like the glue that holds everything together
sections linearly far away are now close
the mediator will take all that information and make sure Pol II goes into the right place and everything happens correctly

Question 13

what are steady state levels?

Answer 13

RNA that is being synthesised in addition to the RNA that is being constantly degraded

Question 14

what is the limitation of steady state levels?

Answer 14

doesnt necessarily tell you what genes are being activated and the frequency of that transcription

Question 15

how can you test what a highly transcribe gene look like?

Answer 15

the idea: put something on the RNA so that when it is being transcribed, there is a handle for which you could identify that particular RNA and then evaluate how much RNA is being made from that given gene
introduce a secondary structure in the 5’ region of the gene
introduce a stem loop, such that there is a protein that recognises that structure
bacteriophage (heterologous) protein that recognises that stem loop, marked with GFP
that stem loop will be formed in every single RNA synthesised
have enhancers and promoters to drive the expression
the GFP will focus, and using computational analysis you can remove all the background GFP

Question 16

in what way does transcription occur?

Answer 16

in bursts

Question 17

how was the transcription of snail during gastrulation investigated?

Answer 17

sna has a very strong enhancer called the shadow enhancer
located 7.5kb downstream of the promoter
make constructs to investigate how transcription is occurring during this period of gastrulation in embryogenesis
3 situations; enhancer deleted, enhancer normal, and enhancer upstream (5’) to assess whether position has an effect
to every situation add 24 SM2 stem loop sites (RNA binding protein will interact with this)
introduce these constructs independently into different fly lines

Question 18

what is transcriptional efficiency correlated with?

Answer 18

correlated with increased “burst” frequency
the stronger the enhancer/more effective transcription is equal to a higher frequency of bursts
works this way in all eukaryotes

Question 19

explain the whole p-granules thing?

Answer 19

started to examine embryos at the very early stages of cell division
all the p granules go to the posterior end of the embryo prior to division
there is a clear segregation
the posterior will give rise to the germ line –> the immortal lineage

Question 20

how do p-granules actually behave?

Answer 20

behave more like liquid liquid condensates (droplets)
in the anterior part, the constituents of those droplets could solubilize, but in the posterior they cannot, so they form condensates
important for almost every reaction that takes place in our cells

Question 21

how can intrinsically disordered domains mediate liquid liquid phase separation?

Answer 21

condensates are also taking place and are important for the transcription reaction
investigators decided to label one of the subunits in the mediator
one of these units, MED1 has an intrinsically disordered region (IDR)
very important for its function
when you introduce mCherry (a way of labelling proteins in vivo)
variant of MED1 that has this IDR into cells with mCherry
forms clumps/puncta
in each puncta there are thousands/millions of copies of MED1
and you can see when you mix it with GFP, GFP doesnt go in
when you mix it with a classic transcriptional activator which also has an IDR
co express them in the same cell type with BRD4-IDR and MED1-IDR
they occupy the same sites
the puncta are actually droplets
like to come together and concentrate, the IDRs enhance/favor those interactions
bring in more till they become large liquid condensates
no longer soluble with rest of nucleoplasm

Question 22

what are some things that influence the formation of those condensates?

Answer 22

places where proteins that would normally come together congregate
formation is dependent on concentration of macromolecules (DNA, RNA, proteins)
the valency of constituents: the electrostatic interactions promote the formation of those condensates
post translational modifications, phosphorylation events and of course the IDRs that are present on given factors
all of those things bring those things together to make condensates

Question 23

how was it showed that the mediator and RNA polymerase II form liquid like structures?

Answer 23

if you label the mediator and label RNA pol II they would from time to time localise, but not all the time
doesnt matter if its with immunofluorescence or in vivo, every once in a while the mediator and RNA pol II come together, presumably activate transcription, and then leave each other and come back and then leave

Question 24

what is the dynamic kissing model?

Answer 24

proteins that form in these loops that require mediator activity and then RNA pol II would form these aggregate like things
condensate, carry out function and then dissociate –> the bursts

Question 25

how are those transcriptional condensates controlled by RNA feedback?

Answer 25

everything comes together: DNA, GTFs, mediatro and RNA pol II
RNA pol II generates a bit of RNA which sticks everything together (positive feedback)
then you create enough RNA so that those electrostatic interactions between RNAs become problematic, and the condensate dissociates