BIO 200 - Molecular Mechanisms of Transcriptional Activation and Repression, Nov 1

What kind of motions do molecules use to move through the cell?

Brownian motion and diffusion

What does the probability that transcription factors will find their target depend on?

Volume they need to explore, how fast they move, size of their target, how large their target is

How fast could a protein the size of a GFP diffuse across the nucleus (and a what speed)?

2 seconds, 20 microm²/s

What trick do transcription factors use to find their target faster?

Combine 1D and 3D motion: they bind nonspecificaly onto DNA then bind until they find their binding site

What technique can we use to track individual copies of transcription factors and study how they find their target?

Single-molecule microscopy

How does a HaloTag work?

Forms a covalent bond with its ligand - ligand can be fused to a fluorescent dye. Can have brighter molecules than GFP.

What are the techniques that cells use to constrain protein diffusion?

• Compartmentalize proteins in organelles (reduces the volume of the search proteins must do)

• TFs organize into subnuclear structures

What are foci?

Proteins that are involved in transcription which co-localize in the nucleus - much larger than a chromatin fiber

Why do foci seem to exist in places where the DNA is less stained?(If staining DNA and tracking TFs.)

DNA stains better when it is chromatin and not transcribing (unwound)

What do foci contain?

Hundreds to thousands of copies of transcription proteins such as BRD4 (TF) or MED1 (part of the mediator complex)

What is the formation of condensates dependent on?

IDR domains in proteins

What are IDRs?

Intrinsically disordered regions

How do IDRs help make condensates?

IDRs in one protein will weakly interact with IDRs in the other condensate - making a transcriptional condensate

Through which process are condensates formed?

Liquid-liquid phase separation

How does liquid-liquid phase separation work for condensates?

AS IDR interactions are transient, but the large number of possible multivalent (each IDR interacting multiple times) - causes the proteins to associate in a liquid-like state

When were LLPs first described in biology?

In C. elegans in structures called P granules which are important during development

Do P granules separate equally during cell division?

No, they all migrate to one side and go to one cell

Can TFs be formed in vitrio?

Yes

What kind of proteins form droplets in vitro?

They must have IDRs

What can transcription condensates contain other than transcription factors?

They contain many other factors and also include RNA pol II that can be used to form a pre-initiation complex

What do condensates be analogous to?

Membrane less organelles as they restrict the diffusion of proteins

What are super-enhancers?

Some genes contain multiple enhancers clustered over a region of 10 or more kb

What can super-enhancers form?

Big transcription condensates

What can the MS2 system be used to study?

Study RN localization

What is the MS2 system obtained from?

An RNA that infects E.Coli

How does the MS2 system work?

Protein MCP binds to an RNA hairpin by making a fluorescent version of MCP we can detect where the labelled RNA is

What is required for MS2 to work?

Multiple copies of the sequence that produces the RNA hairpins have to be introduced in the gene sequence so that it gets transcribed as part of the RNA molecule to be studied

What is an alternative to the MS2 system?

smFISH

What is a drawback of smFISH?

Requires a fixation step, so studied cells are not alive anymore

How does smFISH work?

Fluorescently labelled probed recognize specific sequences on the target RNA molecule

Does transcription occur all at once or in bursts?

In bursts

How many transcription events per burst?

20-100 initiation events

What results in more frequent bursts? Less frequent bursts?

Strong enhancer - sna shadow results in more frequent bursts than that observed with the weaker enhancer (rho NEE) despite similar burst size

What are the bursts thought to result from?

Bursts result from interaction between enhancers and promoter regions

What do transcription bursts represents?

Encounters between enhancers and promoter regions