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)
Describe 5’ capping and its effects
2’ OH is replaced with methyl groups
- enhances mRNA stability (protection from degradation from exoribonucleases)
- required for translation (cap mediates binding of mRNA to the ribosome)
Describe 3’ polyadenylation and its effects
- a lot of adenosine added to the 3’ terminus
- provides stability to protect against 3’ terminus of exoribonucleases
What are 5’ and 3’ mods important in?
Enable mRNA circularization
- mediates ribosome binding
- promotes translational efficiency
- ensures complete processing
IF ribosome is dependent on proper cap/tail mods, then ur ensuring ONLY properly transcribed mRNA is translated
How much the amount of rRNA change in the cell?
Not much, because the cell still needs to make proteins, just different ones depending on diff conditions
T or F: Reverse transcription REQUIRES oligonucleotide primers
True
What are random hexamers?
Random sets of 6 nucleotides in a row
- represent all possible hexamer sequences
- allow reverse transcription of ALL forms of RNA
- bind to any transcript and convert ALL of it to cDNA
What are oligo dT primers?
- typically 5’- (T)18 -3’
- selectively generate cDNA from 3’ polyadenylated mRNA (e.g. wouldn’t generate cDNA from rRNA)
- bias in cDNA library towards the 3’ end of mRNAs (wouldn’t generate a lot of cDNA library for other parts of the mRNA other than the 3’ end)
Describe ribosomal RNA depletion
Allows you to enrich for the RNAs we acc care about (tRNA, mRNA, and long non-coding RNAs) by DEPLETING rRNA
Allows us to look at the whole transcriptome efficiently and doesn’t waste resources on sequencing rRNAs
What is the RNA open reading frame?
A start codon, followed by a long stretch with no stop codon
What are transition mutations
Purine to purine: A –> G
Pyrimidine to pyrimidine: C –> U
What are silent mutations?
No change in AA sequence
What are missense mutations?
Change in a SINGLE amino acid
Can be delirious or non-delirious:
- changing to a similar polarity may not affect
- if this AA changes to for example proline (which is a helix buster) this can significantly damage the secondary structure
What are nonsense mutations?
Insertion of a premature stop codon in the middle of the coding sequence
What is a non-stop mutations?
Loss of a stop codon at the end of the coding sequence
How does the cells deal with mutations?
- cells have capacity to identify and deal with these types of mutations
- mRNA is degraded during the first round of translation in the ribosome
- NO PROTEIN PRODUCED
Describe NMD
Nonsense mediated mRNA decay (decapping and degradation)
- 5’ to 3’ exonuclease
- degraded in first round of translation
- PREMATURE STOP CODONS in final exon trigger NMD
How do we know if its a premature stop and not the actual stop?
When introns are spliced out, and you get an exon-exon boundary, you can get an exon junction where the proteins aggregate around it
if the protein recognizes down-stream protein complexes then it will realize its a PREMATURE stop and not the actual one
How many aminoacyl-tRNA synthetases are required for each amino acid?
ONE IS REQUIRED for each AA
Which one is true (amount of each):
codon > AA
AA > codon
MORE codons than AA’s, kind of obvious
Can ribosomes govern fidelity of charges?
NO only synthetases can
Can ribosomes cover fidelity of codon-anticodon base pairing?
Yes
What are the diff wobble base pairs?
G = U
I = C, U, A
Which site is the tRNA charged with the appropriate AA?
A
P
E
at the A-site
What does each site do:
A
P
E
A site: for aminoacyl tRNA entry, so that the tRNA is charged with the appropriate AA
P site: peptidyl tRNA-growing peptide chain
E site: exit site for uncharged tRNAs
What does the decoding center do? Where is it?
Peptidyl transferase center has a seperate decoding center which reads RNA (proofreading) to ensure proper codon-anticodon pairing
Does the peptidyl transferase center have proofreading capacity?
NO
but there is a decoding center for this
What is the initiator tRNA?
tRNA^fMet
What does tRNA^fMet do?
Interacts with initiation factor 1 (IF1) which is a protein that sheppards the initiation tRNA to the P-site in an assembling ribosome
it does this to the P-site b/c its blocked on the N-terminus (can’t be incorporated in the middle of a growing protein)
How many synthetases charge both tRNA^Met and tRNA^fMet?
ONLY ONE
What it “the eukaryotic tRNA^Met”?
tRNAi^Met
interacts with eukaryotic equivalent of IF2
What is the prokaryotic vs eukaryotic ribosomal binding sequence?
Prokaryotes: Shine-Dalgarno sequence
Eukaryotes: Kozak sequence
Describe Shine-dalgarno
helps recruit the ribosome to the messenger RNA to initiate protein synthesis by aligning start codon with the ribosome P site.
complementary to 16S rRNA
4-9 purines, 8-13 nt upstream of AUG
Why is polycistronic DNA only found in prokaryotes?
Because in eukaryotes splicing of premRNA can occur, but once its mature, one mRNA is responsible for producing ONLY ONE protein in eukaryotes
WHat is polycistronic DNA?
one transcript that can encode multiple proteins
What does prokaryotic IF3 do?
Prevents premature assembly and stabilizes 30S
What does prokaryotic IF1 do?
blocks A-site (ensures a tRNA can’t sneak in there)
What does prokaryotic IF2 do?
chaperones fMet-tRNA^fMet to P-site
What is formed when all the initiation factors are released?
70S initiation complex
What are aminoglycosides?
If codon-anticodon pairing is right it will change conformation
If not right it allows for FLIPPING OUT
IN PROKARYOTES but
at HIGH doses it can read through nonsense mutations in eukaryotes
What is puromycin?
An antibiotic that can block protein synthesis
- structurally resembles the 3’ end of AA of a charged tRNA
- fits into peptidyl transferase center of a ribosome sitting right in the spot where the charged tRNA AA would
- FOOLS ribosome into transferring growing polypeptide chain to it
PREMATURELY TERMINATES ALL TRANSCRIPTION in BOTH EUKARYOTIC AND PROKARYOTIC RIBOSOMES
Is puromycin anchored to the ribosome?
NO not ASSOCIATED or anchored in any way, so as soon as it is attached it just floats away
What are class I vs class II release factors?
Class I release factors (RF-1 and RF-2):
- mimic structure and surface charged of charged tRNA
- fit into A-site and recognize stop codons, allowing release of peptide chain through hydrolysis
Class II release factor (RF-3):
- catalyzes dissociation of RF 1/2 from ribosome
What are the stop codons for tRNAs?
There are no stop codons, there are only release sites
What is negative regulation?
Binding of a repressor protein that prevents or decreases expression
Where does an allosteric effector bind
At a site other than the DNA binding site
What does the lac repressor do?
binds 2 of 3 operator sites, tying DNA in a loop, so RNA pol cannot progress when it binds
What is lac repressor structurally
Hetero-tetramer of two dimers
- same sequence recognized by both dimers
- they are linked up forming the homotetramer
- it makes sense they would recognize the same sequence because they are the same
How can a repressor prevent RNA pol from doing its job?
- blocking binding of RNA pol to promoter
- prevent open –> closed conformational change of RNA pol, which prevents initiation of transcription
- lock RNA pol at the promoter
what does allolactose do?
- allosteric effector
- blocks the ability of the lac repressor to bind the operator
- changes repressor conformation and weakens its affinity for DNA
Do effectors bind DNA themselves or act on proteins that bind DNA?
NOTE: effectors don’t bind DNA themselves, they act on proteins that bind DNA, changing their affinity for DNA thereby changing the actions of the repressor
what is CRP
cAMP receptor protein
- a bacterial ACTIVATOR
- homodimer
- each subunit binds cAMP, and NEEDS cAMP in order to bind DNA
What is cAMP to CRP if CRP requires it to bind DNA?
An effector
What happesn to cAMP when glucose is present?
When glucose is present, breakdown products influence both the production of cAMP and its export from cells
What happens to cAMP when glucose goes up?
When glucose goes up, you get less production and more export of cAMP, and a general reduction in cAMP
this is cuz cAMP is required to drive high expression of Lac operon, if glucose is high you would expect cAMP to be low
Describe activators and repressors
activators or repressors are proteins that have particular DNA binding sites/DNA landing pads within the genome that they latch on to, often influenced by effectors which can regulate that DNA binding, and influence the affinity of the RNA pol for the promoter or its ability to progress beyond the promoter or its ability to change the open/closed conformation and initiate transcription
What are coactivators and corepressors
coacvitvators and corepressors
- don’t bind DNA, just other proteins
interact with other proteins and influence their ability to initiate transcription
difference between a coactivator/corepressor/effector:
- effectors are small molecules that influence the ability of the activator/repressor to bind DNA (cAMP is an effector that influences the ability of the activator to bind DNA in the first place)
- whereas a coactivator serves as a bridge between DNA-binding activator and RNA polymerase (doesn’t bind DNA itself). So you need the activator and the co-activator to initiate the elevated transcription (for the activator to be able to function)
What are long non-coding circular RNAs useful for?
long non-coding circular RNAs can serve as sponges (or distractions) for miRNA
distracting them away from their target mRNA
they share a sequence with the 3’UTR of the mRNA of interest
some of the miRNA will bind this and get sucked up by this, leaving mRNA to be free and bind to proteins
What does miRNA usually bind?
miRNA usually binds 3’UTR of target mRNA and degrades it
