LEC23: Basal & Regulated Transcription Flashcards
what are the phases of transcription?
1) pre-initiation complex formation
2) initiation
3) elongation
4) termination
where/what is the core promoter?
a DNA sequence in immediate vicinity of +1 site
where RNA Pol II binds, directed by other protein factors
may contain “consensus” sequence - similar sequence found in promoter regions of many genes
how are upstream ntds designated from txn start site?
negative numbers
where is the TATA box located re: +1 site?
-20 - -30
what are TFIIs?
transcription factors for RNA Pol II
bind DNA, and each other, to direct localization of RNA Pol II for initiation of txn at +1
what is TFIIH kinase?
a **ser-thr kinase, one **of the transcription factors for RNA pol II
**phosphorylates **the **CTD **of RNA Pol II, & this signals to being transcription
what is the CTD?
C-carboxy-terminal domain of one of the RNA Pol II subunits
has many Ser-Thr residues
gets phosphorylated by TFIIH kinase - **this is the signal to begin transritoin **
what begins txn initiation?
TBP, TATA box binding protein, binding
results in bending of the DNA
what is the signal to begin txn?
phosphorylation of the Ser-Thr residues of the CTD of one of the RNA Pol II subunits
what does the TFIID complex consist of?
TBP and many TAFs, TBP-associated factors, and TFIIs (A, B, D, F, H), and mediator
what is the pre-initiation complex?
complete ensemble of protein complexes at txn initiation sit,
TFIIs, TBP, Mediator, RNA Pol II
what is different between RNA and DNA polymerases?
RNA polymerase can start de novo; DNA polymerase cannot
what needs to happen so that elongation can begin?
PIC formation and binding to the txn initiation site, followed by
promoter clearance
what is the rate of txn elongation?
50 ntd/second
what happens once RNA Pol II complex leaves promoter region to transcribe downstream sequences?
another RNA Pol II complex can load right behind it, begin txn
what do helicase and topoisomerase do?
helicase: enzyme that unwinds the DNA helix; causes tension in helix; part of TFIIH complex
topoisomerase: alters topology of DNA dbl helix by cutting 1 strand of the tightly wound DNA dbl helix, relaxing helix, putting it back together again; relieves tension = crucial for elongation
what happens to phosphates on CTD at termination?
when RNA Pol II comes off the DNA template, a CTD phosphatase takes off the phosphates that were added to the CTD
**de-phosphorylate **when done w/ txn
now RNA Pol II transcribes again
what is the core promoter?
where is it re: +1 site?
what binds to it?
cis element where txn initiation factors bind
-40 to +40, in and around +1 site
TFIIs, mediator bind; but TFIIs may have protein-protein interactions to PIC rather than bind directly to the DNA
what are promoter proximal elements?
where are they?
what do they bind?
cis elements that are bound by unregulated txn factors
-40 to -200, close to core promoter
proteins that constituitively enhance the initiation of txn bind here
what are enhancers?
where are they?
what do they bind?
100-500 bp long cis element of DNA where regulated txn factors bind
can be located anywhere! upstream, downstream, doesn’t matter
multiple txn factors bind to an enhancer to enhance the rate of txn initiation
what % of our genome encodes for txn factors?
10%
what are simple vs. complex enhancer sequences?
simple: multiple repeats of the same sequence, are bound by same txn factors
complex: different DNA boxes w/ different sequences can be bound by different txn factors
what are the elements of a txn factor?
1) DNA binding domain: binds DNA, recognizes sequence; can be a zinc finger or helix-turn-helix
**2) activation domain: **talks to another protein to activate txn initiation rate
**3) dimerization domain: **allows formation of homo- or hetero-dimers (or tetramers) of txn factors
how do txn factors bind?
what are some motif examples?
what does this kind of structure afford txn factors?
as dimers or tetramers
dimers increase txn factor diversity
leucine zipper, helix-loop-helix, zinc finger
why is the # of txn factor complexes that can bind to enhancers and influence rate of txn >>> total # of txn factor proteins?
b/c they can bind as homodimers, tetramers, heterodimers; gives greater combination possibilities
what can the txn factor activation domain interact w/?
1) directly w/ constituents of teh PIC to increase the rate of txn
2) w/ co-activators that change the state of local chromatin
how does it work that enhancers can be very far away from promoter?
DNA loops; not concrete in shape
can have something v. far away, loops back on itself
txn factor binding on enhancer & the PIC as formed at promoter then get brought together
if you had a mutation in TFIID, TBP, RNA Pol II, what would likely be result?
incompatible w/ life b/c so crucial to the basal txn of all genes, cannot form life
where do we often see specific txn factors’ mutations?
diseases
i.e. P53: often mutated in cancer
what enzymes can be recruited to enhancer sequence to modify chromatin?
1) histone modifying enzyme
2) chromatin remodeling complex
what is histone acetyl transferase (HAT)
a histone modifying enzyme that acetylates histone lysine residues
this changes charge from (+) to neutral; reduces interaction btwn histone & DNA
this activity assoc w/ loosening of the histone-DNA interaction which we want for enhanced PIC formaiton, txn initiation
what can reverse acetylation of histones?
HDAC, histone deacetylase
removes acetyl group from histone, restores positive charge to Lysine
restores chromatin structure
usually has inhibitory effect
what is histone methylase, what does it do?
adds methyl group to epsilon amino; methylation of lysine group
changes chromatin confroamtion
can be up or down regulator of gene expression
what does histone demethylase do
removes methyl groups added to chromatin by histone methylase
what does histone kinase do
phosphorylates serine; gives it negative charge
opposite: histone phosphatase
also changes chromatin structure
what does a chromatin remodeling complex do?
changes how nucleosome looks; doesn’t modify histone
what are writers, erasers, readers, re: histone modifications?
writers: enzymes that put on histone marks
erasers: enzymes that take off histones
readers: recognize, modify histone; bind & do something to chromatin structure
what is the histone code?
idea that there is some kind of code that’s read by the cell about state of histones, to either up/down regulate txn
H3 histone modifications:
1) what does methylation of K9 do
2) what does methylation of K4+acetylation of K9 do?
3) what does phosphate on S10 and acetyl on K14 do?
1) heterochromatin formation, gene silencing
2) gene expression
3) gene expression
what do chromatin remodeling complexes do?
change histone protein composition of the nucleosome or reposition nucleosomes on the DNA to expose regulatory sequences
usually involves multiple txn factors in regulation for a single gene
can: subtitue variant histones; move nucleosomes; move histone cores; reveal DNA that now can be accessed by another protein
how many kinds of histones are there
mainly think of H2A, H2B, H3, H4, and H1, but know that **there are different subtypes of histones of the histone core of chromatin **and remodeling complexes can substitute variant histones, change chromatin structure, function
what are epigenetic changes? how are they transmitted?
changes to chromatin that don’t change DNA sequence, but change what chromatin looks like
heritable changes via cell division across generations
how can txn factors’ activity be regulated?
1) post-translational modification (i.e. MAPK pathway: ERK kinase P-lates a txn factor)
2) proteolysis: degredation of a txn factor
3) localization: i.e. NOTCH
4) ligand binding: txn factors need sthg to bind to it, activate
5) synthesis: need a txn factor’s gene to make it!
where/what is a CpG island
high concentration of CG dinucleotide in a small sequence
typially, near promoter sequences
what does DNA methyltransferase do?
what does it do to CpG islands? result?
enzyme that transfers a methyl group to DNA
happens to cytosine of CpG island
causes epigenetic change:
recruits **MeCP2, **methyl CpG binding protein 2: **represses txn **by recruiting a histone deacetylase
how does Rett Syndrome occur?
knock out of 1 copy of MeCP2 gene
MeCP2 is on X chromosome
if have reduced MeCP2 expression, certain genes are expressed at higher level than they should be
