BCH5413 EXAM II
Describe the tree mechanisms that connect DNA methylation with alteration of chromatin structure
1) methylation mark is recognized by MBD. MBD recruits HDAC, HDAC deacetylates local histone tails 2) Methylation mark recognized by MBD. MBD recruits HMT, HMT methylates local histone tails 3) Methylation mark bound by reader protein, reader protein recruits DMNT and further methylates DNA
Explain the three steps in formation of 70s complex
1) once the 30s initiation complex is formed, IF3 comes off allowing a 50s subunit to bind 2)GTP is hydrolyzed after the 50s subunit joins the 30s complex to form the 70s initiation complex - GTP hydrolysis is carried out by the GTPase in the 50S subunit
What are the last three steps of PIC formation?
4) TFIIE Joins and associated with the formed PIC 5) TFIIE Recruits RFIIH to from transcription initiation complex 6) TFIIH has kinase activity and phosphorylates the CTD of RNA pol II which promotes the transition from transcription initiation to elongation
Describe both the similarities and differences between histone acetylation and histone methylation
Acetylation is almost always associated with gene activation while methylation effect depends on its reader HMT are highly specific for histone type and the site to be methylated, HATs are not
Which Histone acetylations are associated with transcriptional activation?
All of them
The amino acid is attached by which kind of bond? Between what is this bond formed?
Amino acid is linked together through a peptide bond. This bond is formed between the carboxyl group of one amino acid and the amino group of another.
What does AAUAA recruit?
CPSF CPSF is therefore not required for the second phase of polyadenylation
Trypsin digestion cleaves the peptides at which amino acids?
Cleaves the c-terminal domain at of lysine and arginine
What does the ITC do?
Cycles through several cycles of abortive initiation, releasing large amounts of 2-3nt long RNA transcripts
What are the prokaryotic initiation factors?
IF3, IF1, IF2
Explain passive demethylation
If DNMT is blocked, methyl marks will be diluted and removed after multiple rounds of replication
What is the function of IF2?
In prokaryotes, it binds to the fmet-tRNA and GTP brings the complex to the p site
With regards to mitochondrial import pathway Where does the protein folding occur?
In the mitochondrial matrix
What are the functions of the 5' cap?
Protect from degradation stimulate splicing CAP binding proteins facilitate transport of mRNA out of the nucleus Stimulate polyadenylation decapping involved in regulation of mRNA turnover
What is the function of a histone reader? Give an example of how the reader can chance the outcome of a histone mark
Readers recognize post translational modifications and bind to them-- this recruits or stabalizes various transcription factors. A reader can recognize multiple marks and marks can be recognized by multiple readers ex. bromodomains recognize acetylated histones and bind them, their catalytic domain then acetylates neighboring histone tails creating a feed forward process ex. HPA chromodomain binds to H2K9Me. HP1 likes to interact with this mark. If multiple HP1 bind to marks, they interact with each other and create a closed heterochrmatin
How is DNA copied at replication fork - Continuous, Discontinuous or Semi Discontinuous?
Semi discontinuous
What is phosphorylated in relation to pol II C terminal domain (CTD)?
Serine 5 and Serine 2
What are the two phases of polyadenylation?
Slow Phase- PAP has to grab onto something that dosnt have a and start adding 10-12 As. Creates a landing pad for poly a binding proteins Rapid phase- poly a binding protein adds As rapidly (typical tail 100-250nts)
The GET complex (Get1,Get2 and Get3) mediate what kind of protein translocation? And in which organelle?
Transmembrane translocation protein is translated in cytoplasm. GET3 uses ATP, binds to get1/2 comles in membrane, translocate protein to membrane, uses atp for hydrolysis ER membrane
T/F Nucleosome remodeling complexes facilitate the binding of transcription factors
True
Glycosylation usually takes place in which amino acid?
Usually ASN usually have sugars attached usually occurs in membranes
Mitochondrial signal peptidase requires which metal co factor?
Zinc
With regards to mitochondrial import pathway What are the two major requirements?
* Chaperone ATP HSC70 *Binding to import receptor Tom20/22 *Matrix chaperone ATP
State three possible mechanisms by which miRNA repress Translation?
* Compete for cap binding and repress initiation * compete for elF6 and prevent ribosome assembly * promote degredation, decapping de adenylation *cause ribosome dropoff, inhibit translation post initiation
Name some of the complexities of eukaryotic transcription when compared to prokaryotic transcription
* Eukaryotic transcription involves decondensing chromatin * The three main polymerases in eukaryotic systems are not sequence specific. They require transcription factors to recognize promoter sequence for them. In prokaryotes, te sigma factors are sequence specific to -10 region in the promoter * In eukaryotes, transcription and translation are compartmentalized and divided by the nucleus, transcripts need to be transported to cytoplasm upon creation * Eukaryotic organisms form pre-mRNA and then further process it to make mature mRNA
How did the linker scanning experiment demonstrate that the TATA box is required for transcription while the GC boxes are not?
* mutated regions throughout the TK promoter - Regions mutated within the TATA box destroyed promoter activity - regions mutated in GC box regions affected transcription efficiency but did not abolish it.
In general overview, what are the steps necessary for transition from the PIC to the EEC?
0) initiation- formation of the PIC- initially transcribing complex (itc)- includes abortive initiation 1) promoter clearance/ escape- early elongation complex (EEC)- promoter escape complete, nascent RNA stabiliy associates with transcription complex 2) promoter proximal pausing- initially characterized in MYC gene but now found genome wide 3) Productive elongation transcription elongation through nucleosomes
Explain two ways in which DNA methylation in promoters can silence gene expression
1) Methylated CpG is bound by methylated CpG binding proteins. These have a domain that repress transcription or they can recruit additional proteins that repress transcription 2) the methylated CpG forms a structure that blocks transcription factors from binding
What are the steps necessary for transition from EEC to productive elongation?
1) PTEFB phosphorylates DSIF, NELF, and SERII of pol II. CTD stimulated productive elongation 2) NELF dissociates from the transcription complex. DSIF, TFIIS, and PTEFB track with pol II along the gene 3) TFIIF, ELL, and elongin stimulate pol II elongation activity and associate with the elongation complex
What are the different models of chromatin remodeling complexes?
1) SWI/SNF 2)ISWI 3)INO80/SWR1 4)Nurd * all are ATP dependent
Name the two models for PIC assembly. What is the one similarity between the two?
1) Sequential assembly pathway 2) RNA polymerase holoenzyme pathway Promoter recognition occurs by TFIID in both
What are the first three steps of pre-initiation complex formation (PIC formation)?
1) TBP (a subunit of TFIID) recognizes and binds the TATA box 2) TFIIB binds and complexes with TFIID at the promoter 3)Recruitment of RNA pol via association with TFIIF
What are the subunits of 70s and 80s ribosomes?
70s ribosome= prokaryotes · 30s subunit-decodes mRNA · 50s subunit- links amino acids together through peptide bonds 80s Ribosome- eukaryotes · 40s · 60s
What CTD phosphorylation happens first and what does it do
CTD phosphorylation of ser5 helps recruit the capping enzymes
What method would you use to show that two distant regions of DNA interact to enhance transcription?
Chromatin configuration capture (3C) Cross link the cell to create a covalent interaction that holds together the looped out region of the enhancer and core transcriptional elements. Use restriction enzyme to cut the DNA held in this association Ligate the two strands in this DNA together dissolve cross linking design primer that would match the expected sequence for these two regions of DNA to be interacting joining together after ligation. If the regions were interacting and ligated together, you will see PCR product
The core of DNA Pol III consists of ___________________
DNA polymerase, 3-5' exonuclease, stimulates epsilon exonuclease A, E, O
T/F What is the function of DNA B and DNA G at E.coli replication?
DNAB is the helicase DNA G is the primase
How is DNA methylation maintained after DNA replication?
DNMT1 is a writer for maintenance of methylation it uses hemimethylated DNA as a template to methylate the unmethylated strand. Because DNA replication is semi conservative, it recognized the methyl mark on the conserved strand and uses it as a template to methylate the nascent strand.
Compare the early spliceosome complex to the A complex
Early complex- sr proteins identify exonic and splice enhancers U1snRNP binds 5' splice site u2af binds polypyrimidic segment to identify the branch point by U2AF the A complex forms when u2snrnp is recruited to the branch point by U2AF
T/F Aminoacylation of tRNAs requires GTP
False, it requires ATP protein synthesis requires GTP
T/F Wobble allows the different aminoacyl-tRNA to pair with same codon
False, this is backwards. Wobble allows the same aminoacyl tRNA to pair with multiple codons
T/F Ligase enzyme joins the amino acids to the respective tRNAs
False, this is done by aminoacyl trna synthetases
T/F RNA primers are 5-15kb long
False- 10-12nt
T/F Fmet-tRNA is the initator tRNA in Eukaryotes
False- it is in prokaryotes
T/F DNAsel and RNAsel are examples of nucleosome remodeling complexes
False- these are enzymes that degrade regions of DNA or RNA not bound by chromatin, they do not remodel the chromatin though
T/F SWI/SNF can act only as negative regulation of transcription
False- this family works by sliding the nucleosomes or ejecting them. This allows for easier access to the chromatin which can aid in either activation or repression
Compare group 1 and group 2 self splicing introns
Group 1: The imitating event is attack by an independent guanine molecule Group 2: The initiating event is intramolecular attack by A residue of an intron to form a lariat
Expand IRES? What is it made of?
Internal ribosome entry site allows cap independent initiation they are made of RNA requires an AU rich tract, a special structure, and initiation factors elf2, elf4, and other proteins
What is the function of TFIIB
It contributes to promoter selection, recruitment of TFIIF association with RNA pol II
How does DNAsel hypersentivity assay work?
It degrades linker DNA
what is the function of Klenow fragment?
It is the portion of DNA pol I responsible for 3'-5' synthesis and proofreading
How does lysine acetylation on the histone tail alter the interactions between nucleosomes?
It weakens the electrostatic interactions between the amino tail of H4 and the acidic region of H2A/H2B dimer, which prevents the formation of compact 30nm fibers
What is the structure of the 5' Cap?
Its a guanine nucleotide connected to the mRNA by a 5' to 5' triphospate linkage. Guanine is methylated on the 7 position
What is the function of the mediator?
Just like it sounds 1) transmits signals from transcription factors to polymerase 2) functions as a coactivator 3) is shown to interact with the CTD of RNA Pol II 4) required for transcription activation
I have a drug X which is an inhibitor of MASKIN. Explain how the addition of the drug X affect translation?
MASKIN= repressor protein binds to eLF4E and CPEB when bound to eLF4E, it cant bind to elF4G, so translation is inhibited. If you inhibit MASKIN, elfE is free to bind with 4g and translation can occur
Explain active demethylation
No enzyme that can cut the c-c bond demethylation occurs through modification of the group instead TET methylcytosine dioxygenase oxidizes the methyl group. In presence of ATP can go through successive oxidation steps possible that there is an enzyme that can cut the group once it has been oxidized but has not been found yet
How does polio virus inhibit translation?
Polio virus has a viral protease that cleaves off the n-terminal domain of elF4g. When cleaved, it cannot recognize caps and capped cellular mRNA is no longer translated
What is the function of Drosha?
Recognizes hairpin, processess pre-miRNA, 2-base 3' protruding end, end recognized by exportin 5
What is the function of Dicer?
Recognizes pre-miRNA processess yielding a 21-23 bp dsRNA fragment 2- base 3' protruding ends
How does S1 nuclease protection assay work
S1 nuclease degrades singe stranded nucleotide chains. Add a primer for the full length of the transcript being probed. It will be degraded to match the size of the transcript that it binds to. If it has been cleaved, you will see a reduction in the length of the primer
What is the function of TFIID
TBP+13 TAFS recognize and bind TATA Box and other core promoter elements, Helos to confiugre DNA to RNA POL II surface General transcription factor contains the TATA box binding proteins, TBP association factors (TAFs) it nucleates PIC assembly though TBP binding to TATA sequence or TAF binding to other promoter sequences
Explain why the lane for construct 1471 has three bands
There are two poly A sites, so you have two possible products and 1 product for the labeled primer
How are DNMTs related to DNA methylation machinery?
These are the DNA methyltransferases. They add methyl marks to DNA and maintain them during replication
T/F Okazaki fragments are 1-2 kb long on prokaryotes
True
T/F Protein synthesis requires GTP
True
T/F all of the nucleosome remodeling complex work in an ATP dependent manner
True
Which snRNAP binds to the 5' splice site?
U1snRNP
Describe an experiment to show evidence for translocation channel
Use a free ribosome with a message- attach a florescent dye on the lysine group- the dye can be quenched by iodine In the cytosol, the dye can be quenched so you dont get a signal If a tight seal is formed with the membrane, dye is not quenched and you get a signal if you insert iodine in the lumen of the ER and there is a tight seal, the dye can be quenched and you do not get a signal again
Describe evidence that some RNAs have catalytic activity for splicing
Use in vitro synthesized u2 and u6 snRNA fragments plus a oligonucleotide containing a branch point with no splice site. The branchpoint A attacked the phosphodiester bond in U6 itself, forming a branched oligonucleoide
what causing strong pausing or transcript slippage for promoter escape?
a 5 nt long nascent RNA collides with the Bfinger of TFIIB stress causes collapse of transcription buble- B finger is ejected and TFIIB is released-- marks transition to EEC
Indexing during translation mainly requires____________ .
all three initiation factors as shown by toe printing experiment
What is the function of Ago
binds to ss miRNA strand selection based on thermodynamic asymmetry forming miRNP inhibits translation of HAND2 and other muscle mRNA it helps guide miRNA and siRNA to target mRNA
What is the function of IF3?
binds to the 30s subunit to keep is separated from the large subunit
What is the function of IF1
binds to the a site and keeps the fmet tRNA from binding there
Describe an experiment to show Translation and translocation occur simultaneously
cell free protein synthesis without microsomes--> completed proteins with signal sequences- dont get incorporated into the microsomes and are degraded when you add proteases Cell free protein synthesis with microsomes Cotanslational transport of protein into microsome and removal of signal sequence. Mature protein chain without signal sequence, protected by microsome and not degraded when you add protease
What happens when Kozak sequence is not strong?
if the consensus sequence is weak, it may require additional factors or features in the mRNA to recognize the imitation site may use leaky scanning may cause formationof upstream mini cistrong- strong sequence but followed by a stop codon a short sequence away
Describe any three ways by which lncRNAs target gene regulation?
long noncoding RNA RNA-DNA interaction (direct sequence interaction) RNA-RNA interaction- interact and cause conformation change RNA-Protein interaction-creates a bridge and loops out region between transcription factors, prevent repressors from binding to gene miRNA sponge
List three post translational protein modifications
methylation carboxylation acetylation phosphorylation
Explain why the lane for 1474 only had two bands
one of the poly A sites was mutated so there is only one product and a product for the labeled primer
What CTD phosphorylation step happens second and what does it do
phosphorylation of the serine 2 in the body of the gene. Helps establish transition to elongation
Example of a protein that is carboxylated
prothrombin- binds ca2+ helps to initiate clotting mechanism
What is the function of topoisomerase?
relieving strain in the DNA ahead of the replication fork
Function of DNA Polymerase Gamma is ___________________
replication of mitochondrial DNA
What advantage does shRNA have over siRNA?
siRNAs are transiently expressed in cells shRNAs can be stably integrated through virus-mediated transduction *Ability to use viral vectors for delivery to overcome the difficulty of transfecting certain cell types, *he option to control shRNA expression using inducible promoters, * the ability to co-express them with a reporter gene. Additionally, they may cause fewer off-target effects
What is the main function of SRP-Receptor
signal recognition particle receptor PARTICLE NOT PROTEIN SRP- Stops protein synthesis and directs proteins to RER SRPreceptor- protein in the RER that binds to SRP and releases the hold on translation
how is abortive initiation reduced in ITC?
the B finger of TFIIB and a switch domain of pol II stabilize short RNA
With regards to mitochondrial import pathway Where is the sequence location in the protein?
the N terminus and is cleaved off after import
The U2snRNP is recruited to _________ by _________
the branch point by u2af
Which subunit does peptide bond formation occur in?
the large subunit 50s in prokaryotes, 60s in eukaryotes
How are MBDs related to DNA methylation Machinery?
they are the methyl CpG binding proteins- they are responsible for reading and interpreting DNA methylation marks
Define Wobble
this refers to the observation that the third base of acodon is allowed to move slightly from its normal position to form a non-watson crick base pair with the anticodon this allows the same aminoacyl trna to bind to more than one codon
What is the function of hydrolysis of GTP after 70s initiation complex is formed?
to release IF2 and GDP from the complex so the polypeptide chain elongation can begin
How many transesterification reactions are required for one splicing event
two
Describe the experiment that proves that DNA replication at the fork is semi discontinuous
use heavy hydrogen to label DNA administer in varying pulses of Heavy hydrogen - demonstrate that short fragments of DNA are being generated with short pulses -demonstrate that long fragments of DNA are being formed with long pulses -repeat with mutant ligase- long DNA does not form anymore