Transcription Activity in Eukaryotes

Question 1

what are the 2 types of chromatin and which of these is transcriptionally active?

Answer 1

heterochromatin - not transc active, vry condensed

euchromatin - is transc active, less condensed

Question 2

what is the experiment that can be done to test DNase hypersensivity?

Answer 2

immature reticulocytes that dont express globin gene are used
their chromatin extracted
it’s digested with DNase
the resulting DNA extracted and put on a gel
the globin gene can be radioactivally marked and visuallised
= only see a single band

repeart with mature reticulocytes
that DO express the globin gene
= you’ll see 2 bands (which if you add up their molecular weight, it’’s the same as the single band from before)

see diagram in onenote

Question 3

what does this experiement show

Answer 3

the difference between the 2 is that the structure of chromatin had chnaged from transcriptional inactiviy (in immature cell) to transcriptional activity (in mature cell)

cuz
DNase, in order to cut the DNA, has to find a way through the chromatin
and cuz it was able to do so easier in the second time
it shows that chromatin structure has changed to allow easier access to DNA

Question 4

explain the structure of a histone

Answer 4

non-polar globular domain
N - terminal variable arm
C - terminal variable arm

these arms are +vely charged cuz they contain amino acids (lysine & arginine)

Question 5

how do nucleosomes interact with DNA?

Answer 5

via electrostatic interactions
+ve arms from the histone protein stick out and interact w/ -ve backbone of DNA

Question 6

What are the 3 modifications histones can go through

Answer 6

methylation
Acetylation
Phosphorylation

Question 7

Methylation as modification of histones, and what mechanisms it’s involved in

Answer 7

CH3 group added to lysine and histidine residues

Involved in
gene activation and repression
Dna replication

Question 8

Acetylation as modification of histones, and what mechanisms it’s involved in

Answer 8

addition of CH3CO2- (-ve) group to lysines of the core histone

Involved in
Nucleosome assembly
Gene activation

Question 9

Phosphorylation as modification of histones, and what mechanisms it’s involved in

Answer 9

addition of phosphate group to lysines of H1

Involved in cell division
Transcription
Chromatin structure

Thats all u need to know for this one

Question 10

What sequence can you get upstream of a promoter sequence?

Answer 10

Enhancer sequence
Acts as a set of binding sites for specific transcrpton factors can bind to

Question 11

How are enhancer sequences diff from promoter sequences in terms of where they exist on the gene

Answer 11

Promoter seq can only exist near the start site and facing the right direction

enhancer sewuences can be placed almost anywhere (can be vry far from promoter sequence, upstream, downstream, etc)
(some enhancers are naturally downstream from the promoter sequence, but they can be put there articially as well jus fyi)

Question 12

How are enhancer sequences diff from promoter sequences in terms of orientation

Answer 12

promoter has to be in a fixed orientation

enhancer sequence is position and orientation indepedent
so baiscally
can be swapped, put it somewehre else and it’ll still work

Question 13

once proteins have bound to the enhancer sequence, what can they interact with?

Answer 13

RNA polymerase and general transc factors

Question 14

what 2 domains do all transc factors need to have in order to interact properly

Answer 14

a DNA binding domain - recognises the dna sequence so it can bind

transcription activation domain - recognises polymerasej and the rest of the transcription apparatus

Question 15

see onenote for notes on the interactions between promoters and enhancers - basal transcription apparatus

Answer 15

:)

Question 16

what do all these basal proteins between the promoter and enhancer sequences allow for?

Answer 16

long distance interactions - DNA bending and looping
- remember, enhancers can be vry far from promoters

allows general factors and polymerases to be recruited

allows HATs and chromatin modifier complexes to be recruited

Question 17

describe the pathway that allows extracellular stimuli to nucleus to change gene transcription

Answer 17

ligand interaction with receptor on the plasma membrane

this leads to generation of a 2nd messenger small molecule (e.g. cAMP) (this is amplification because single hormone + single receptor = lots of small molecule)

this allows activation of protein kinase

allows regulation of transcription factor activity

Question 18

what changes can take place when phsophorylation occurs

Answer 18

changes in charge (phophate = negative

this could change structure

or how protein interacts with other proteins and with DNA

Question 19

how can phosphorylation affect biological activity of a transcription factor

Answer 19

its ability to act with DNA and polymerases

stability of protein
(ie how long it survives for)

Question 20

JAK Stat1 pathway: - what signalling molecule is important for cell cytotoxity responses

Answer 20

inteferon y (gamma)

Question 21

whats the name of the kinase protein that the inteferon y receptor is coupled with?

Answer 21

JAK kinase

Question 22

what does the JAK kinase directly phosphorylate?

Answer 22

Stat1 proteins (a transcription factor)

Question 23

what does stat1 exist as usually in the cell

Answer 23

as unphosphorylated monomers

Question 24

what happens to the stat1 monomers when they are phosphorylated

Answer 24

dimerise
this can then translocate to the nucles and interact with promoters and enhancers in the gene

see onenote for this whole process

Question 25

CREB pathway: how does adrenaline lead to the production of cAMP in signal transduction pathway

Answer 25

adrenaline binds to its receptor - adenylate cyclase (an enzyme)

this enzyme sythesizes the converstion of ATP to cAMP

cAMP is the 2nd messenger which acts as amplicfication signal

Question 26

what kinase protein does cAMP activate

Answer 26

protein kinase A

Question 27

what does protein kinase A do

Answer 27

translocates to nucleus

phsophorylates CREB (already bound to DNA in the nucleus)

Question 28

what does the phosphorylation of CREB do?

Answer 28

changes its abilty to interact with the coactivator protein called CBP

Question 29

what is CBP and what does it do

Answer 29

Creb Binding Protein
gets recruited to the promoter

this allows transcription as it is a histone acetylase protein

see onenote for this whole pathway

Question 30

protein family- what are nuclear hormone receptors and some examples

Answer 30

they’re ligand dependent transcription factors /receptors
not bound to membrane but are acc inside the cell

estrogen receptors
thyroid hormone receptors
vit D receptors
corticosteroid

Question 31

what is something all the ligands for nuclear horomone receptors have in common?

Answer 31

all are hydrophobic molecules
easily diffus throguh plasma memb

Question 32

sub families of nuclear hormone receptors: homodimers?

Answer 32

2 receptors of the same type which bind together and interact with DNA

Question 33

sub families of nuclear hormone receptors: heterodimers

Answer 33

2 receptors that are different that dimerise together

Question 34

sub families of nuclear hormone receptors: monomers

Answer 34

just singular one
rare

Question 35

corticosteroid/glucocorticoid pathway: what are the receptors in the cytoplasm bound to and what does this do

Answer 35

receptors are bound to chaperone proteins
these inhibit the receptors

so until the steroid hormone comes to interact with the glucocorticoid receptors, they remain inactive and as monomers in the cytoplasm

Question 36

corticosteroid/glucocorticoid pathway: what does binding of the steroid hormone to the receptor cause

Answer 36

dimerisation of the glucocorticoid receptors
(homodimerisation)
these are now activated and translocate to the nucleus

Question 37

corticosteroid/glucocorticoid pathway: what does the glucocorticoid dimer do after it’s translocated to nucleus?

Answer 37

binds to promoter
activates transcription

Question 38

what is the molecule that acts as a ‘dimerisation surface’

Answer 38

Zinc fingers

Zn complexed w/ amino acids
basically allow the receptor to be dimerised

Question 39

what does a polycistronic mRNA do

Answer 39

can code for more than one polypeptide