15: Molecular Basis of Transcriptional Regulation

what allows cells to differ despite them all having the same DNA? what does that mean?

Differential Gene EXPRESSION

not all genes are transcribed in every cell

what genes get transcribed determine what cell it becomes

another word for gene expression is

transcription DNA—> RNA

do all cells of the same organ have the same expression or can they differ? ex. do all hepatocytes express the same genes

NO external signals can change gene expression under different conditions

Controlled Gene Expression:

what are the different levels of control that occur at each step?

1. DNA —> RNA

2. RNA —> mRNA

3. mRNA (nucleus) —> mRNA (cytosol)

4. mRNA —> inactive RNA

5. mRNA —> protein

6. protein —> active protein

7. protein —> inactive protein

1. transcriptional

2. RNA processing

3. RNA transport and localization

4. mRNA degradation

5. translational

6. protein activity

7. protein degradation

what are some examples of CIS regulatory sequences?

enhancers and promoters

where are enhancers and promotors found in relation to the coding sequence? what are they called?

enhancers: both upstream (5’) and downstream (3’)

promotors: upstream (5”)

CIS REGULATORY SEUENCES

what is the difference between promotors, enhancers, and transcription factors?

promoters and enhancers are sequences OF DNA that the transcription factors can BIND to stimulate or repress gene expression

Transcription Regulation on Bacteria:

___________ has a SINGLE circular DNA molecule about 4.6 X 10-6 bp

• only SOME of its _______ proteins are turned on at any given time

• is transcription independent of environment factors?

• e. coli

• 4300

• NO may be modulated

what do operons do in bacteria?

list an example.

regulate transcription of clusters of genes

tryptophan operon includes 5 genes to encode for different enzymes needed for its synthesis

this cluster of genes are all transcribed at the same time when tryptophan levels are low

• The tryptophan operon has _______ promotor

  '

• what regulates the operon in case there is too much tryptophan? what is this process called?

• one

• tryptophan repressor

• NEGATIVE regulation

how are tryptophan repressors activated from their inactivated state?

when tryptophan levels are high tryptophan will bind to the repressors changing their conformation allowing them to bind to the operator within the promotor

once repressor bound RNA polymerase can no longer bind

what is a transcription activator and why is it needed?

can transcription occur with just the activator?

binds to the operator allowing RNA polymerase to bind TIGHTLY

• sometimes the activator must also bind to a metabolite like sugar

which operon in bacteria codes for proteins required to import and digest lactose?

lac operon

when is the lac operon transcribed and why?

when glucose levels are low and lactose is available because lactose is the second option for energy when glucose isn’t available

what is the state of the repressor in each situation?

• absence of lactose

• presence of lactose

• absence of glucose

• absence of glucose and presence of lactose

• repressor bound to promotor

• repressor can no longer bind to the promotor

• CAP protein binds to the promotor (but if the repressor is still on it won't activate transcription)

• CAP binds to promotor and activates transcription (NO REPRESSOR bc/ there is lactose)

what is the first gene of the lac operon that allows it to break down lactose? which enzyme does it encode?

gene = LacZ

enzyme = b-galactosidase

what 2 names does CAP go by? when is it activated?

cAMP receptor protein or Catabolite Activator Protein

• activated when there is low glucose since low glucose activates adenylyl cyclase —> increased cAMP

are cis regulator regions always found close to the activation site in eukaryotes? what happens if they are far away?

NO some cis regulatory regions can be farther away from the activation site

a transcription factor bound to a cis regulator region loops the DNA attaching to the RNA polymerase allowing it to tighten its bond to DNA and then returns back

the ______ _________ region refers to the whole region of DNA involved in controlling the expression of the gene

gene control

what are co-activators and co-repressors?

bind to repressors and activators not DNA directly to activate and inhibit gene transcription

transcription activators can work synergistically, what does that mean?

if there is only one activator there is less transcription than if there were two

more units of transcription = more of that protein

how can transcription regulators (factors) be regulated?

1. expression of the gene that encodes for them

2. translation of the protein

3. translocation of the protein into the nucleus

how can transcription activators enhance transcription?

1. Promotor Looping

2. RNA polymerase II Recruitment

3. Elongation Facilitation

4. Modification of Chromatin

Transcription Activation:

• Promotor Looping: activators bound to _____________ facilitate looping bringing the enhancers closer to the _______. This can help _________ and _____________ the transcription machinery

• _________ ____________ Recruitment: some activators can help recruit ______ ________ to the ____________ facilitating transcription initiation

• _____________ facilitation: some activators help promote the transition from ___________ to __________ increasing transcription efficiency

• Modification of ______________ structure

• enhancers promotor recruit stabilize

• RNA polymerase II promotor

• elongation initiation elongation

• chromatin

what issue can arise with the nucleosomes (8 histones) within the chromatin structure?

hard for RNA polymerase II to bind to and transcribe DNA into RNA

what are the 7 ways gene expression can be blocked?

1. competitively bind to DNA

2. mask activation surface

3. compete with transcription factors

4. methylate the promotor

5. chromatin remodeling

6. histone deacetylases

7. histone methyl transferase

methylation of _______ within the _____ rich regions in the ____________ can suppress transcription of the gene

cytosine CpG (cytosine next to guanine) promotors

acetyl groups on histones are added typically to which region? what does acylation do? how about deacetylation?

lysine

acetylation = chromosome less dense so transcription factor and RNA Polymerase can bind

deacetytation: chromosome condense harder for transcription factors to bind

how are histones deacytelated? does this lead to activation or repression of transcription?

HDAC (eraser) histone deacytelase

deactivation

how does methylating histones “silence” them?

proteins containing MBD will bind to the methylated CpG regions

MBD= methyl CpG Binding Domain

SIN3A binds to proteins with MBD which then recruits the eraser HDAC which takes off acetyl groups from lysines which will cause chromatin to become condensed and unable to be transcribed

what are the 5 proteins with a methyl CpG binding domain (MBD)? what binds to THEM? what happens once they are bound to?

MECP2 and MBD1, 2, 3, 4

SIN3A

SIN3A recruits HDAC to take off acetate from lysine and make chromatin condensed repressing transcription c

would it be possible to turn a hepatocyte into a neuron?

YES if you transcribed genes that encoded for neurons and repressed genes that encoded for hepatocytes in a liver cell than it would turn into a neuron

how were scientists able to have an eye grow on the leg of the drosophilia fruit fly?

activated the gene for eyes (eyeless gene) on the leg cells

do activators have to bind directly to their cis-regulatory sequences (promoters)

NO coactivators can activate other transcription factors to activate their target gene

you can have a NETWORK of genes to express one gene

what is a master regulatory transcription factor?

activators that activate networks of coactivators

TF1

how does a differentiated cell remain differentiated?

cell memory

daughter cells produce transcription regulators through positive feedback

what regulates the master gene? how? why is this essential?

master gene regulates itself!

this is possible through positive feedback (master gene regulates other transcription factors) those factors then promote master gene

Master gene will be passed down to daughter cell!

what is an example of a transcription factor that binds directly to the cis-regulatory site and activates gene transcription

MyoD

transcription factors can regulate themselves (autocrine) and each other through both __________ and _________ _____________ _____________

negative and positive feedback loops

how does the notch pathway relate to differentiation?

when a ligand binds to notch it can either cause

• differentiation of cardiac and bile duct cells

• excessive proliferation without differentiation if overactivated

Notch inhibitors/agonists are under investigation for restoring differentiation in some cancers

inhibitors

Some genes are transcribed transiently

what does that mean?

they are transcribed when needed due to environmental changes

what is an example of transient transcription? what makes it possible?

when glucocorticoid hormone is released many genes are transcribed to deal with environmental stress

glucocorticoid + receptor complex acts as transcription factor

What do prednisone, dexamethasone, and hydrocortisone do?

bind to glucocorticoid receptors which will then translocate to the nucleus and bind to genes as a transcription factor activating transcription

SUPPRESS INFLAMMATION

CAN BE USED DURING TRANSPLANTATION

upon growth factor stimulation signaling pathways such as the __________ pathway can lead to regulation of ___________ and consequently the regulation of specific genes that can respond to stimulus

MAP Kinase transcription factors

which is an example of a gene that is upregulated in response to growth factor stimulus?

cyclin D

how do growth factors impact suppression of Rb (tumor suppressor)?

growth factors stimulate cyclin D which binds to Cyclin dependent kinase and together they phosphorylate Rb unblocking transcription factors (E2F and DP1)

what does estrogen do?

estrogen binds to intracellular estrogen receptor where it dimerizes and works as a transcription factor for genes dealing with preganacy, ect.

can genes only be altered at the transcription level?

NO changes may be made AFTER transcription = posttranscriptional

1. DNA Transcription STARTS —>

2. Possible Attenuation —>

3. ______________ —>

4. _______________ —→

5. possible RNA covalent modification and RNA editing→

6. ___________ ________________—>

7. spatial localization in the _____________ —>

8. start _________________—>

9. possible translational recoding —>

10. possible RNA ___________—→

11. Continued Protein Synthesis

• capping

• splicing

• nuclear export

• cytoplasm

• translation

• stabilization

transcription of some genes is inhibited by premature termination of transcription

transcriptional ATTENUATION

what causes terminal attenuation?

RNA chain adopts a structure that causes it to interact with RNA polymerase in a way that causes the transcription to stop

when protein is made regulatory proteins bind to RNA and reverse attenuation

can attenuation be reversed?

YES after protein is made regulatory proteins bind to RNA and reverse attenuation

MOSTLY PROKARYOTES AND VIRUSES (HIV)

short RNA sequences that can interfere with RNA polymerase

Riboswitches

do riboswitches turn RNA polymerase on or off?

ON if there isn’t a small molecule (metabolite) attached to it

OFF if a small molecule is attached to the riboswitch

riboswitches are more seen in _____________

what is an example of one in ___________?

prokaryotes

glycine riboswitch — controls gene involved in glycine metabolism

although riboswitches are more prevalent in prokaryotes what is an example of a eukaryotic riboswitch?

Thiamine Pyrophosphate (TPP) Riboswitch- regulates gene expression involved in thiamine biosynthesis uptake

__________ can produce different proteins or different forms of a protein from the same gene

splicing

CDD4 is a transmembrane glycoprotein involved in

when CDD4 is spliced it can produced different proteins that can …

• adhesion migration and signaling

• promote migration, invasion (can be cancerous), and differentiation or inhibit migration

COVALENT modification of mRNA that alters its nucleotide sequence and thus changes its encoded message

RNA editing

what are some examples of RNA editing?

deamination of

adenine to inosine

cytosine to uracil

RNA editing within the coding region can lead to changes in the

• amino acid sequence

• premature termination

• truncated protein

edits made OUTSIDE the coding region can impact

splicing or transport out of the nucleus

__________is a nucleotide only found in RNA

how is it formed?

Inosine

deamination of adenosine (RNA edit)

The RNA edit of Adenosine to Inosine is able to regulate which gene?

GluA2 gene (subunit of AMPA)

AMPA = excitatory ion channel (Na+ and K+) in the brain allowing action potentials to go through

A- I editing changes glutamine to arginine NORMAL

what happens due to a lack of A→ I editing in GLUA2?

unedited GLUA2 is permeable to Ca2+ leading to neuronal damage specifically temporal lobe epilepsy

Apolipoprotein B exists in both an edited and unedited isoform

What edit is made?

In which organ are there edits and in which organ are there no edits?

Cytosine —> Uracil

liver = no edit

intestine = edit

what does the unedited Apolipoprotein B do in the liver?

what does the C-U edited Apolipoprotein B do in the gut (intestine)?

liver= secretion of VLDL

gut = transport dietary lipids

Altering the time in which mRNA leaves the nucleus is another form of regulation

When does the mRNA typically leave the nucleus?

after introns and incompletely processed RNAs are degraded

does the location of the mRNA in the cytosol once it leaves the nucleus matter ?

YES

mRNA is usually located close to where the protein will be needed

the mRNA that encodes for a protein that will be secreted out of the cell is located near the ER (sequence on N terminus determine location)

do all eukaryotic RNA have similar stability?

what is the average half-life of mRNAs?

what is the half-life of Beta-Globulin?

no, it varies

less than 30 minutes

more than 10 hours

Do genes for growth factors and transcription factors typically have long or short half-lives?

How is RNA destroyed?

SHORT

Poly A shortening, de-capping, degradation through 3’ Poly A end

what is the first step of mRNA destruction?

what two events happen simultaneously after the first step

poly A tail shortening (reduced about 25 nucleotides)

• De-capping of 5’ end —> degradation of exposed RNA

• further degradation through 3’ poly-A end

while MOST mRNAs are subject to both degradation of Poly A tail and 5’ cap certain _______ ______ impact the efficiency of degradation

the half life of mRNA is also effected by how efficiently it is __________________

Any machinery that increases translation efficiency will ___________ degradation

• nucleotide sequences

• translated

• reduce