Transcription Activity in Eukaryotes Flashcards
what are the 2 types of chromatin and which of these is transcriptionally active?
heterochromatin - not transc active, vry condensed
euchromatin - is transc active, less condensed
what is the experiment that can be done to test DNase hypersensivity?
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
what does this experiement show
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
explain the structure of a histone
non-polar globular domain
N - terminal variable arm
C - terminal variable arm
these arms are +vely charged cuz they contain amino acids (lysine & arginine)
how do nucleosomes interact with DNA?
via electrostatic interactions
+ve arms from the histone protein stick out and interact w/ -ve backbone of DNA
What are the 3 modifications histones can go through
methylation
Acetylation
Phosphorylation
Methylation as modification of histones, and what mechanisms it’s involved in
CH3 group added to lysine and histidine residues
Involved in
gene activation and repression
Dna replication
Acetylation as modification of histones, and what mechanisms it’s involved in
addition of CH3CO2- (-ve) group to lysines of the core histone
Involved in
Nucleosome assembly
Gene activation
Phosphorylation as modification of histones, and what mechanisms it’s involved in
addition of phosphate group to lysines of H1
Involved in cell division
Transcription
Chromatin structure
Thats all u need to know for this one
What sequence can you get upstream of a promoter sequence?
Enhancer sequence
Acts as a set of binding sites for specific transcrpton factors can bind to
How are enhancer sequences diff from promoter sequences in terms of where they exist on the gene
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)
How are enhancer sequences diff from promoter sequences in terms of orientation
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
once proteins have bound to the enhancer sequence, what can they interact with?
RNA polymerase and general transc factors
what 2 domains do all transc factors need to have in order to interact properly
a DNA binding domain - recognises the dna sequence so it can bind
transcription activation domain - recognises polymerasej and the rest of the transcription apparatus
see onenote for notes on the interactions between promoters and enhancers - basal transcription apparatus
:)
what do all these basal proteins between the promoter and enhancer sequences allow for?
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
describe the pathway that allows extracellular stimuli to nucleus to change gene transcription
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
what changes can take place when phsophorylation occurs
changes in charge (phophate = negative
this could change structure
or how protein interacts with other proteins and with DNA
how can phosphorylation affect biological activity of a transcription factor
its ability to act with DNA and polymerases
stability of protein
(ie how long it survives for)
JAK Stat1 pathway: - what signalling molecule is important for cell cytotoxity responses
inteferon y (gamma)
whats the name of the kinase protein that the inteferon y receptor is coupled with?
JAK kinase
what does the JAK kinase directly phosphorylate?
Stat1 proteins (a transcription factor)
what does stat1 exist as usually in the cell
as unphosphorylated monomers
what happens to the stat1 monomers when they are phosphorylated
dimerise
this can then translocate to the nucles and interact with promoters and enhancers in the gene
see onenote for this whole process
CREB pathway: how does adrenaline lead to the production of cAMP in signal transduction pathway
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
what kinase protein does cAMP activate
protein kinase A
what does protein kinase A do
translocates to nucleus
phsophorylates CREB (already bound to DNA in the nucleus)
what does the phosphorylation of CREB do?
changes its abilty to interact with the coactivator protein called CBP
what is CBP and what does it do
Creb Binding Protein
gets recruited to the promoter
this allows transcription as it is a histone acetylase protein
see onenote for this whole pathway
protein family- what are nuclear hormone receptors and some examples
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
what is something all the ligands for nuclear horomone receptors have in common?
all are hydrophobic molecules
easily diffus throguh plasma memb
sub families of nuclear hormone receptors: homodimers?
2 receptors of the same type which bind together and interact with DNA
sub families of nuclear hormone receptors: heterodimers
2 receptors that are different that dimerise together
sub families of nuclear hormone receptors: monomers
just singular one
rare
corticosteroid/glucocorticoid pathway: what are the receptors in the cytoplasm bound to and what does this do
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
corticosteroid/glucocorticoid pathway: what does binding of the steroid hormone to the receptor cause
dimerisation of the glucocorticoid receptors
(homodimerisation)
these are now activated and translocate to the nucleus
corticosteroid/glucocorticoid pathway: what does the glucocorticoid dimer do after it’s translocated to nucleus?
binds to promoter
activates transcription
what is the molecule that acts as a ‘dimerisation surface’
Zinc fingers
Zn complexed w/ amino acids
basically allow the receptor to be dimerised
what does a polycistronic mRNA do
can code for more than one polypeptide
