Mod 4 short Flashcards
what are changes in gene function that do NOT involve changes in DNA sequence?
- Chromatin remodeling
- Histone modification
etc
(sequence is the same, but the degree to which it is expressed is altered)
What does acetylation of lysine residues generally do?
Generally ENHANCES transcription
(neutralizes positive charge, loosening inter-nucleosome interactions therefore loosening compaction and allowing it to be more available)
What does methylation of Lysine and Arginine residues generally do?
- recruits histone-binding protein complexes
- stabilizes either “open” or “closed” state through this recruitment
- can act to enhance or repress transcription depending on the situation
- NO NET CHANGE in charge
H3K9ac
a mark of activation
H3K4me3
a mark of activation
H3K9me3
a mark of repression
What are the groups involved in reversible modification of N-terminal histone tails?
- Histone acetyltransferases (HATs)
- Histone deacetylases (HDACs)
- Histone methyltransferases (HMTs)
- Jumonji family (KDMS)
What do HATS do?
Histone acetyltransferases
- transfer acetyl groups to histones
- loosens it up and makes it more accessible to other proteins
- associated w/ euchromatin (active)
What are HDACs and what do they do?
Histone deacetylases
- remove acetyls from tails
- closes off transcription
What are HMTs and what do they do?
Histone methyltransferases
- H3K9me3 and K3K7me3 –> associated with heterochromatin state (NOT ACTIVE STATE)
- other ones associated with euchromatin
What are KDMs and what do they do?
Jumonji family
- removes methylation of histone tails (histone demethylases)
What are cis vs trans histone modifications?
cis: modifications that directly impact the chromatin structure
e.g. lysine acetylation (adding acetyl group to lysine weakens the group and can open up the closed part of the genome)
trans: recruits other proteins that enact chromatin change
What are bromodomains?
Protein motifs that recognize and bind acetylated lysines
- it acetylates nearby histones, propagating the open state thus INCREASE in gene expression
What are chromodomains?
Proteins motifs that bind methylated lysines
- can stabilize either open or closed state (can promote either activation or repression)
- H3K9Me3 is associated with REPRESSION
What is phosphorylation in respect to histones?
A histone modification
- adds a negative charge to the histone tail
- serine, threonine and tyrosine can be phosphorylated
- an important intermediate step in other histone mods
(initiates recruitment/release of histone modifying enzymes or chromatin remodelling complexes
Can multiple mods happen to one histone?
Yes, and they ultimately result in activation/repression of gene activity
What are chaperones?
Acidic proteins that are needed for nucleosome assembly
(need a neg charge to neutralize the positive charge of histones so that they can bind)
Difference between dNTP, rNTP, ddNTP, etc
Provide some differences in transcription vs translation
- rNTPS in transcription, tRNAs in translation
- selective (duplicate entire genome in DNA replication, vs transcription u pick the parts you’re transcribing based on environmental needs)
- no primers
- only one strand used as a template
Which ones are complementary, and which ones are similar?
Coding
Template
RNA
coding (nontemplate) DNA is complementary to the template DNA
coding DNA is similar to RNA except that RNA has U instead of T
What is the bacterial RNA polymerase made up of?
RNA holoenzyme is made up of 5 subunit core enzyme + a sigma factor co-enzyme subunit
What does the sigma factor co-enzyme subunit do?
Gives selectivity to bind particular regions of the genome and not others
What happens when we incorporate the incorrect nucleotide in regards of RNA Pol?
RNA Pol slows down or “stalls”
If it doesn’t, we get this fraying end that causes RNA pol to backtrack (peels inappropriate RNA off the DNA template into rNTP entry channel and the catalytic core has some level of intrinsic nuclease activity “nucleolytic proof reading”)
What is alpha aminitin? What does it do?
blocks active site of eukaryotic RNA pol II
- this inhibits mRNA production
- other RNA pols not affected (doesn’t affect tRNA and rRNA)
what is the eukaryote equivalent of sigma factors?
Transcription factors (TFs)
What do transcription factors do?
- lead RNA pol to a particular part of the genome that needs to be transcribed (allows for selective binding of RNA pol to core promoter of a particular gene)
Steps of transcription
- Transcription factors lead RNA pol to core promoter of particular gene
- binding to closed complex where the double helix is still attached
- formation of 17 nt transcription bubble (opening up of DNA; “open complex”)
- abortative initiation (primer-free initiation of transcription)
- elongation
- termination and recycling
Describe primer independent transcription initiation
- transcription starts just by having the two correct rNTPS beside each other, making a phosphodiester bond between them
- REALLY UNSTABLE (“abortative transcription”)
- if you move past 8-10 nucleotides it becomes stable
True or false: Each pol (1,2, and 3) requires unique TF
True
True or false: ALL transcription factors require the TATA binding protein (TBP)
True
Where does the TBP bind?
The minor groove
Preferentially binds an AT region because 2 H-bonds is easier to latch on and crack open
What does TBP do?
Inserts Phe between base pairs, which can distort or crack open the double helix
Essential to the transcription of ALL genes; including those lacking a TATA box in their promoter
Where is the TATA box normally found
30 bp upstream
Do you using denaturing on non-denaturing gel for each of the below:
- EMSA
- DNA footprinting assay
EMSA:
NON-denaturing (keeps covalent interactions (DNA-protein) in place)
DNA Footprinting Assay:
DENATURING gel (because using a non-denaturing gel would bias our results because its bound to protein)
In prokaryotes, mRNA is degraded when in respect to translation?
BEFORE transcription is even terminated (no introns cut out, no mods on ends, etc)
Its just translated and immediately subject to degradation
Is pre-mRNA subject to modifications in:
- eukaryotes
- prokaryotes
NOT in prokaryotes
Yes in eukaryotes
When is pre-mRNA subject to degradation in eukaryotes?
Newly transcribed mRNA is subject to mods IMMEDIATELY prior to exit from nucleus
What are some mods for pre-mRNA?
- introns cut out (splicing), end mods (5’ cap and 3’ poly-A tail), nucleotide editing
- important for stability in translation
When are mods removed?
After we export the mRNA from the nucleus we get translation and then we remove the mods (to make the transcript susceptible to degradation)