Lecture 31

Question 1

How can transcription of the Lac operon be enhanced

Answer 1

In conditions of high glucose, the glucose inhibited the conversion of ATP to cAMP, so theres no cAMP to bind CAP
In conditions of low glucose, theres nothing to inhibit the conversion of ATP to cAMP, so it happens, so cAMP complexes with CAP (catabolite activator protein) and this complex sits just upstream of the Lac operon, binding at site P (promoter) and facilitating the attachement of RNA polymerases to transcribe the Lac operon so the cell can have another available mechanism to get energy

Question 2

Where is the CAP site

Answer 2

CAP site is just upstream of the RNA polymerase binding site, like just upstream of the -35 landmark
This is helpful because the CAP protein interacts well with the polymerase and well as the CAP site, so it’ll promote its attachment

Question 3

Will we have Lac operon mRNA if glucose is present but lactose not

Answer 3

No, if theres glucose present then well have low cAMP so not binding to CAP so no activation of transcription
If theres no lactose theres nothing to inhibit the Lac operon repressor so it continues repressing, so no lac operon mRNA

Question 4

Will we see lac operon mRNA if theres glucose present and lactose present

Answer 4

If theres glucose present we have low cAMP meaning low CAP-cAMP meaning low activation of transcription
If theres lactose then theres at least some inhibition of the repressor going on, so there will be some transcription because prok default is to transcribe, so we get a little mRNA

Question 5

Will there be Lac operon mRNA if there no glucose but lactose present

Answer 5

No glucose means high ATP-> cAMP conversion, high binding of cAMP to CAP so we get activation of transcription of Lac operon
To top it off theres lactose present so it can inhibit the repressor so that also helps with activation of transcription
So yes a good amount of Lac operon mRNAs

Question 6

What exactly is chromatin

Answer 6

DNA and its associated proteins, wrapped up around histone octamers and into nucleosomes, its non naked and loosely packages DNA

Question 7

What are some examples of heterochromatin

Answer 7

Telomeres, centromeres, repetitive elements, anything non-transcribable, could be structural (like when interacting with nuclear lamina)

Question 8

What’s the main difference between prokaryotic and eukaryotic transcription

Answer 8

Prokarytic DNA is much more accessible and is default in the on state
Eukaryotic DNA is bundled up tight, even when its in its resting state so default is off, many more things have to happen before transcription can occur

Question 9

Enhancers

Answer 9

Sequences that promote transcription, they make it more efficient for a given gene
They’re distance independent, cis acting, can be near or far from the transcriptional start site, can be up or down stream, can even be in introns
For its enhancing function to happen the right protein must bind to it, TF

Question 10

Silencers

Answer 10

Many of the same properties as enhancers but ultimately represses transcription for a given gene
For its repressive function to actually happen a protein must bind to it, TF

Question 11

How specifically do enhancers promote transcription

Answer 11

They interact with transcription machinery (like RNA polymerases), either directly or indirectly
They influence chromatin structure, like how condensed it is, by loosening it they’d be making it more accessible

Question 12

What’s the difference between a TF and a cofactor

Answer 12

TFs have the ability to bind DNA directly and cofactors dont
Note that other than that they can do most of the same things, activation or repression domains for transcription machinery, domains for dimerization, ligand binding, etc
Cofactors are often recruited to DNA by TFs to help out

Question 13

What’s an enhanceosome

Answer 13

Its basically a complex of the TFs, RNA pol 2 and whatever cofactors are required assembled at an enhancer which will be spatially close to the gene in question due to all the folds and twists in DNA

Question 14

What’s the yeast Gal system

Answer 14

System by which eukaryotic yeast cells only transcribe the machinery required to digest galactose when theres galactose present (waste otherwise), the need to transcribe the enzymes (4) and such to convert galactose to glucose such that it can be used for energy metabolism

Question 15

What enzyme activates the transcription of the Gal system

Answer 15

Gal 4 binds to to enhancers called UAS, upstream activators sequences, theres a UAS before each Gal gene required for the system, gal 4 is a transcriptional activator

Question 16

If we have GAL4 mutants can we still digest galactose

Answer 16

No because without this transcriptional activator we cannot transcribe the required genes, no proteins to do the converting and digesting

Question 17

What does Gal80 do

Answer 17

It’s a regulatory protein of the Gal system in yeast, it sits on GAL4 which is transcriptional activator such that it cannot do its job of initiating transcription
So Gal80 prevents transcription

Question 18

Will the Gal system be active if theres no Gal80

Answer 18

Yes, it’ll actually be constitutively active, it will not turn off because Gal80 was repressing the activating function of GAL4, no inhibition= constitutive activation

Question 19

What does gal3 do

Answer 19

Gal 3 is a regulatory enzyme in the Gal system
It senses the presence or absence of galactose, in the presence of galactose and ATP, gal3 changes conformation such that it can now remove Gal80 from GAL4, inhibiting the inhibitor in the presence of the substance needing to be degraded

Question 20

If gal3 is non-functional can we have a functional gal system

Answer 20

No, if theres no gal3 theres nothing to inhibit gal80 (the inhibitor), if you cant inhibit the inhibitor all you’ll get is inhibition of the system, so no transcription

Question 21

GAL4

Answer 21

Regulatory protein in the gal system, binds DNA as a transcriptional activator
It does its thing by recruiting RNA pol 2 to the site (mediators, stuff like that) as well as chromatin modifying proteins (like to unwind and open chromatin in preparation for transcription, makes it more accessible)

Question 22

How do you test whether theres enhancer sequences in a given DNA

Answer 22

You use the reporter gene assay
You take all your potential enhancers, make different constructs out of them, include the promoter region as well as a reporter gene, something that when expressed you’ll see if clearly, like GFP, to quantify activity of enhancer
You take these constructs and introduce them to different cells and quantify the GFP levels, compare them, and if there’s one with exceptionally high levels its likely an enhancer that was promoting transcription
Note: you base expression off of what the control gives you, control being just promoter and reporter gene assay

Question 23

What are insulators

Answer 23

Specific proteins that block enhancers to make sure theyre only enhancing their gene, required for proper gene regulation
They help the DNA form into loops such that the cis regulatory elements are in proximity to the gene they must act on, these loops can also serve to distance enhancers from the wrong promoters (if theyre far enough they wont recruit transcriptional machinery to it)

Question 24

TADs

Answer 24

Topological associated domains
Formed by insulators in nucleus, theyre basically chromosomal arrangements that ensure enhancers and silencers only act on their designated genes
It’s a physical loop

Question 25

Chromatin remodelling

Answer 25

Chromatin may need to be remodelled for transcription because its often tightly wound up into nucleosomes and therefore inaccessible to the transcription machinery Swi/Snf complex of coactivators has to come in, nudge the nucleosome aside such that that binding site can be exposed and transcritpion initiated

Question 26

Histone modifications

Answer 26

Histones can be modified on their N terminus, like tails sticking out, they can be ubiquitinated, phosphorylated, acetylated, or methylated, each one having a different effect on DNA compaction

Question 27

By who are histones modified

Answer 27

They can be modified by writers, the writers add the modifications like acetylation or methylation The same modifications can be removed by erasers, histone code erasers These modifications are recognized by histone code readers, and once recognized we can have recruitment of relevant proteins

Question 28

What effect does acetylation have on histones and DNA in general

Answer 28

Acetylation happens on lysines and the net effect is a relaxation of the chromatin such that it unwinds enough to read it, and the better you can read it the more transcription you’ll get Note that the acetylation itself neutralizes the positive charge on lysines, minimizing its interaction with DNA, it also creates binding sites for histone code proteins that promote activation

Question 29

Writers and erasers for acetylation

Answer 29

Writers add teh modifications, so acetyl is added to lysine via HATs, histone acetyltransferases Erasers remove the modifications, so acetyl is removed from lysine via HDAC, histone de-acetylates

Question 30

Histone methylation

Answer 30

Happens to lysines and arginines by histone mytheltransferases, HMTs The lysine side chain can be methylated multiple times, each signifying something different in the histone code Many of these methylations are associated with gene silencing

Question 31

H3K9Me

Answer 31

Histone 3 lysine 9 methylation promotes heterochromatin formation HP-1, the readers, recognizes H3K9 Me, the binding of multiple Hp1s compacts the chromatin, promoting heterochromatin formation while also increasing activity of HMTs, like a positive feedback loop until its closed

Question 32

How do we stop heterochromatin compaction by methylation if its a spreading kind of process

Answer 32

We have barrier insulators that kind of latch and block further propagation of the compaction These insulators are bound to HATs that add acetyls and loosen DNA, so it stops the spread and promotes the expansion from that point on