Exam 3 Flashcards
What are assays that determine RNA secondary structures?
RNAse Digestion
DMS-seq
SHAPE-seq
What are the assays that determine RNA abundance?
(q) RT-PCR
PT-PCR
RNA-seq
What is the assay that determines promoter/consensus sequences?
CHIP-seq
What is the assay that determines rate of transcription?
GRO-seq
What are the 3 enzymes used in RNAse digestion assay and what do they do?
RNAse V1: cleaves dsRNA
RNAse 1: cleaves ssRNA
RNAse T: cleaves ssRNA
If you were using RNAse V1 to determine the secondary structure what would you see on the gel if the temperature was high?
nothing: RNAse V1cleaves dsRNA so it would all be denatured
If you were using RNAse 1 to determine the secondary structure what would you see on the gel if the temperature was high?
everything: RNAse 1 cleaves ssRNA with would be all of the RNA at high temps
What is the modification of DMS-seq?
modify A and C in ssRNA
Is DMS-seq genome wide or sensitive?
genome wide
What is special about DMS-seq?
it can be done in vivo and in vitro
In DMS-seq where does the RT polymerase stop?
at all the modifications of the A and Cs
Are there DMS signals present at dsRNA in DMS-seq charts?
no; it only deals with ssRNA
DMS signals in DMS-seq represent what?
ssRNA
What is the most accurate assay at predicting RNA secondary structures?
SHAPE-seq
What is the modification of SHAPE-seq?
2’ OH on ssRNA
What is special about SHAPE-seq that makes it so specific?
single nucleotide resolution (observes each base)
SHAPE-seq and DMS-seq have the same mechanism but what 2 things are different about them?
- SHAPE-seq has single nucleotide resolution (more specific)
- different modifications
What RNA abundance assays are relatively quantitative?
(q) RT-PCR
RNA-seq
(q) RT-PCR and RT-PCR are the same except for what 2 things…
(q) RT-PCR is quantitative and measures every cycle while RT-PCR is not
(q) RT-PCR and RT-PCR is genome wide or sensitive?
sensitive
What RNA abundance assay uses fluorescence?
(q) RT-PCR
What is the equation in (q)RT-PCR to determine the fold of a sample?
2 ^ (cycle # - cycle #)
What RNA abundance assay is the most commonly used?
RNA-seq
Is RNA-seq genome wide or sensitive?
genome-wide
What assay can detect intron splicing?
RNA-seq
Why do you need the whole genome sequenced for RNA-seq?
you must be able to match the sequenced fragments to their location in the genome (aids in identification of splice sites)
What are the dips in RNA-seq charts represent?
introns/splicing
Is CHIP-seq genome wide or sensitive?
genome-wide
What does CHIP-seq use to tag RNA?
antibodies
what does CHIP-seq idenitfy?
location of proteins that bind to DNA (like sigma factor)
Why do you need the whole genome sequenced for CHIP-seq?
you need to be able to match the sequenced fragments that had the protein bound to it to its location in the genome
CHIP-seq chart shows letters at positions in the genome; what does this help identify?
consensus sequence
What is the modification in GRO-seq?
changing UTP to BrUTP
What does GRO-seq use to tag BrUTP?
antibodies
What assays use antibody tagging?
GRO-seq and CHIP-seq
Is GRO-seq genome wide or sensitive?
sensitive
What assay looks at strands being actively transcribed by RNA polymerase?
GRO-seq
What assay needs different adaptors added to each end of fragments and why?
GRO-seq
needed to identify the direction of transcription
What does the first positive peak in GRO-seq graphs show?
proximal pausing
What 3 things did GRO-seq discover?
- proximal pausing
- bi-direction transcription
- transcription (bi-directional) at enhancers even though they don’t code for anything
What 3 things are special to RNA making it less stable?
- 2’ OH
- single stranded
- uracil (incorrect base pairing)
How many hydrogen bonds does G-C, A-U and G-U have?
G-C: 3
A-U: 2
G-U: 2
Out of G-C, G-U and A-U what is their strength ranking?
G-C (strongest)
A-U
G-U (weakest)
What is the most common unusual base in RNA?
pseudouridine
unusual bases are most common in what RNAs?
tRNAs
What is the purpose of unusual bases in RNA?
increase stability
What RNA assays are sensitive and not genome wide
(q)RT-PCR
GRO-seq
What does Non-Watson-Crick base pairing triple helixes do to RNA?
increase stability
What is the difference between an internal loop or multi-loop in RNA secondary structure?
multi-loop has more than 2 branches coming off it
What is different about A form RNA?
shorter and fatter
What is an example of a G-quadruplex?
GGGG (telomeres)
Are secondary or tertiary RNA structures more stable?
secondary (lower free E)
What proves structure = function in RNA?
ncRNA secondary structure are more conserved than their sequences
______________ are ribonucleoproteins which guide RNA molecules to their appropriate destinations
signal recognition particles (SRP)
eukaryotic SRPs are made of _____ RNA
7SL
What is pRNA?
packaging RNA
What are ribozymes?
catalytic RNA
What do natural ribozymes do?
formation of peptide bonds
cleave phosphodiester bonds
What do artificial ribozymes do?
glycosidic bond formation
RNA phosphorylation
What happens if RNA is a ribozyme AND a riboswitch?
1 sequence with 2 structures with different functions
What are riboswitches?
sensitive to environmental changes
What are aptamers?
RNA that binds to specific ligands
What is special about RNA in the RNA v. Lipid world that show they were first?
store genetic information and catalytic reactions
What were the first enzymes?
ribozymes
what RNA was the first that lead to life?
self-replicating RNA
What is transcription?
RNA synthesized from DNA template
what performs transcription polymerization?
RNA polymerase
What are 3 similarities between RNA and DNA polymerase?
require a template
goes in 5’–3’
adds single nucleotides at a time
Does RNA polymerase require a primer?
No (DNA polymerase does)
Can transcription happen outside of S phase in prokaryotes?
yes
Is RNA or DNA polymerase more error prone?
RNA polymerase
Are transcription and translation coupled in eukaryotes?
No (it is in prokaryotes)
How many RNA polymerases does bacteria have?
1
How many subunits does bacterial RNA polymerase have?
5
What is the prokaryotic holoenzyme of RNA polymerase?
the active form of RNA polymerase (sigma factor bound)
What is not always attached to prokaryotic RNA polymerase?
sigma factor
What does the prokaryotic RNA polymerase sigma factor do?
directs polymerase to promoter
Do all prokaryotes have the same number of sigma factors?
No all different
Does bacterial RNA polymerase have a helicase?
No, uses Mg2+ isomerase to unwind DNA
What are the 4 steps of bacterial transcription initation?
- RNA pol sigma factor recognizes and binds to promoter (closed complex)
- DNA winds by Mg2+ isomerazation (open complex) transcription bubble
- first phosphodiester bond formed (unstable ternary complex)
- release of sigma factor (stable ternary complex)
0
0
Do sigma factors have to bind to exact consensus sequences?
No, they can vary slightly
Why is it beneficial for bacteria to have different variations of consensus sequences?
allows different levels of that genes expression and different times
The more the consensus sequence matches the original…
the stronger the binding of the sigma factor = more transcription
0
0
What specifically on the promoter does the sigma factor recognize?
consensus sequence
The _____ domain on bacterial RNA polymerase binds the UP element, thereby strengthening RNA Pol’s binding affinity to the promoter
alpha - CTD
Upstream promoters are present on ________ expressed genes
highly expressed genes
What does DNA footprinting identify?
RNA polymerase binding site (sigma factor)
What is abortive initiation in bacteria?
RNA polymerizes a few times but then it is stuck because sigma factor is still attaching it to promoter
What is promoter escape?
in abortive initiation polymerase is able to escape and polymerize because sigma factor released
in bacteria, _________ forms ahead and behind transcription bubble during elongation
positive supercoils
In bacteria, is transcription a smooth process?
No (proximal pausing)
What stalls RNA polymerase in transcription in bacteria?
strong GC stem loop
In bacterial Intrinsic termination, what causes the RNA polymerase to fall off and release transcript?
UUUUUUU
How does Rho-dependent termination in bacteria happen?
GC stem loop stalls polymerase, Rho factor catches up to polymerase, Rho melts off the transcript and polymerase
Why is post-transcriptional modification in bacteria rare?
transcription and translation coupled
What is a post-transcriptional modification in bacteria?
polyadenylation
Polyadenylation in bacteria _______ the mRNA while in eukaryotes it _______ it
bacteria - destabilizes
eukaryotes stabilizes it
All rRNA is derived from a single precursor in bacteria. Why is this possible?
rRNA is not translated into a protein so it has time to be modified into different types of rRNA
What degrades bacterial RNA?
endosomes and exosomes
Why is the polyA tail added to bacterial mRNA?
bacteria have hairpins at the end of mRNA so polyA allows exonucleases to degrade mRNA
What is constitutive gene expression?
expressed all the time
What is repressible gene expression?
normally off
What is inducible gene expression?
normally on
What regulation is most important in bacteria?
transcription regulation
What kind of system is Lac operon?
inducible – negative and positive regulation
What Lac gene makes the repressor?
Lac I
What happens to cAMP levels when lactose is present?
high
What happens to CAP when lactose is present?
bound
What happens to RNA polymerase when lactose is present?
binds to promoter
What happens to Lac genes when lactose is present?
transcribed
What happens to Lac repressor when lactose is present?
allolactose blocks binding site so it doesn’t bind to operator
What does Lac Z transcribe?
B-gala
What does Lac Y transcribe?
perm
What does Lac A transcribe?
transA
What happens to cAMP levels lactose is absent?
low
What happens to CAP when lactose is absent?
not bound
What happens to RNA polymerase when lactose is absent?
not bound
What happens to Lac genes when lactose is absent?
not transcribed
What happens to Lac repressor when lactose is absent?
binds to operator
Do does cAMP bind to CAP when lactose is present?
yes
Does cAMP bind to CAP when lactose is absent?
no
In the presence of glucose and no lactose look like what?
the same cycle as no lactose present
In the presence of glucose AND lactose, what happens to CAP?
not bound
In the presence of glucose AND lactose, what happens to the level of cAMP?
low
In the presence of glucose AND lactose, what happens to the repressor?
not bound
In the presence of glucose AND lactose, what happens to RNA polymerase?
weakly binds
In the presence of glucose AND lactose, what happens to the Lac genes?
little transcription
What type of system is Trp?
repressible system with 2 negative regulations
When Trp is high in the cell, what happens to the repressor?
Trp binds to repressor and they bind to operator
When Trp is high in cell, what happens to RNA polymerase?
not bound
What happens to Trp genes when Trp is high in cell?
no transcribed
When Trp is low in the cell, what happens to the repressor?
there is no Trp to bind to repressor, so repressor doesn’t bind
When Trp is low in cell, what happens to RNA polymerase?
binds
What happens to Trp genes when Trp is low in cell?
transcribed
What is the second line of defense for Trp operon?
attenuation control
When Trp levels are high, what happens to the leader sequence?
hairpin loop is formed = no transcription
When Trp levels are high in a cell, what happens to the level of Trp-tRNA?
increase
When Trp levels are low in a cell does the polymerase stall at the leader sequence or pass through?
stall
What are riboswitches?
RNA that changes its secondary structure
Riboswitches are common in ___________
bacteria
sRNA bind ____________ to mRNA
complementary
one __RNA can regulate multiple target genes
sRNA (small RNA)
What sequence allows attenuation control of Trp operon?
leader sequence (can adopt 2 structures)
What does RNA polymerase I in eukaryotes transcribe?
rRNA
What does RNA polymerase II in eukaryotes transcribe?
mRNA
snoRNA
miRNA/siRNA
sRNA
lncRNA
What does RNA polymerase III in eukaryotes transcribe?
tRNA
sRNA
How many subunits does eukaryotic RNA polymerase II have?
12
How are eukaryotic II and prokaryotic RNA polymerases similar
structure
What assays use antibody tagging?
CHIP-seq
GRO-seq
What does transcription factor TFIID do in eukaryotic RNA polymerase II?
recognize TATA box (promoter)
What does transcription factor TFIIH do in eukaryotic RNA polymerase II?
unwinds DNA and releases polymerase from promoter (phosphorylation of CTD tail)
Is there a universal promoter sequence for eukaryotes?
no
Where are core promoters?
close to start site
Where are proximal promoters?
distant from start site
Where are distal promoters?
far from start site
How do eukaryotes fine tune levels of expression using promoters?
combinations of proximal and core promoters
How are proximal promoters used even though they far not near start site?
DNA folds and uses a mediator (loops form; neighborhood)
TFII___ binds to TATA box via the _____
TFIID
TBP
TFIIH has ______ activity and ________ CTD causing the release of mediator allowing polymerase to move
helicase
phosphorylation
What transcription factor in eukaryotes is important for promoter selection?
TFIID
TBP binding to promoter causes __________ in DNA making unwinding easier
bend
What are the 4 steps of eukaryotic transcription initation?
- TFIID bind to TATA box
- TFIIB is recruited to TATA box
- RNA polymerase recruited with TFIIF
- TFIIE and TFIIH join complex = complete pre-initiation complex
Eukaryotic transcription elongation requires…
elongation factors
eukaryotic RNA polymerase always does proximal ________
pausing
When does 7mGTP capping first happen in eukaryotes?
elongation
When does splicing start in eukaryotes?
elongation
What are the 3 ways nucleosomes are dealt with in eukaryotes?
- chaperons displace nucleosomes
- polymerase can move around them
- co-transcriptional modification of histones can displace them
What are the co-transcriptional modifications in eukaryotes?
5’ capping
splicing
polyadenylation
Explain the CTD cycle?
CTD contain a repeat with Serines that get phosphorylated and will recruit certain factors during transcription
What does Ser5-P do in transcription of eukaryotes?
recruits capping and splicing factors
What does Ser2-P do in transcription of eukaryotes?
recruits polyadenylation and termination factors
When is Ser5-P most abundant in eukaryotic transcription?
initiation/elongation because it recruits capping and splicing factors
When is Ser2-P most abundant in eukaryotic transcription?
elongation/termination because it recruits polyadenylation and termination factors
What facilitates co-transcriptional modifications in eukaryotic transcription?
CTD
What is the first transcription modification to be done in eukaryotes?
7-methylG capping
Are 5’ caps added to all RNA polymerase II transcripts in eukaryotes?
yes
What kind of bond is made in 5’ capping?
5’-5’ phosphodiester
Why are 5’-5’ phosphodiester bonds used in 5’ capping?
resistant to exonucleases
________ group is added to the 2nd and 3rd nucleotides after the 5’ cap
methyl group
5’ capping aids in the first ________
intron splicing
Are polyA tails added to all RNA polymerase II transcripts?
no
Does intron splicing in eukaryotes have to be precise?
yes; it could cause a frameshift
are there more intron or exons?
introns
What mainly does the splicing on introns in eukaryotes?
spliceosomes
In major intron splicing in eukaryotes, what is recognized at the 5’ end and 3’ end?
5’ – GU (T)
3’ – AG
In minor intron splicing in eukaryotes, what is recognized at the 5’ end and 3’ end?
5’ – AU (T)
3’ – AC
Exon and intron boundaries are marked by ______
CTD
What are the steps of the spliceosome process?
- A (branch point) is pushed out of sequence by snRNA
- A pairs with G farther up the sequence (beginning of intron) forming a loop
- loop is removed (intron)
- splice site is marked by exon junction complex (EJC)
What is different about group II self-splicing compared to normal splicing?
same as regular but there is no spliceosome complex
What is different about group I self-splicing compared to normal splicing?
uses free G instead of A = no loop
no complex
Why do mammals have alternative splicing forms?
allows for diversity of the function of 1 mRNA (can be tissue specific)
What are the 3 options of transcription termination in eukaryotes?
- allosteric model
- torpedo model
- combination
How does the transcription termination allosteric model work in eukaryotes?
RNA poly II is destabilized by the conformational change of sequence and falls off
How does the transcription termination torpedo model work in eukaryotes?
exonuclease degrades RNA from the 5’ end and displaces RNA polymerase II
What does the post-transcriptional base-editing cause?
change in protein sequence
Base editing in eukaryotes in most common in what type of RNAs?
tRNAs
What is A to I base editing done by?
ADARs
A to I base editing is involved in ______ processing
miRNA
What is A to I base editing important for?
nervous system and development
What is C to U base editing done by?
APOBECs
mRNA methylation is the only modification that is ____________
transient (reversible)
in mRNA methylation, what are readers?
recognize and bind to modified bases
in mRNA methylation, what are writers?
modify bases
in mRNA methylation, what are erasers?
reverse modification
What are the 3 examples of mRNA quality control in eukaryotes?
- non-stop decay
- no-go decay
- nonsense-mediated decay
When is the eukaryotic mRNA quality control non-stop decay recruited?
recruited when mRNA doesn’t have a stop codon
When is the eukaryotic mRNA quality control no-go decay recruited?
when ribosome starts translation then stalls because an intron is not removed
When is the eukaryotic mRNA quality control nonsense-mediated decay recruited?
when stop codon is added before the real stop codon
Decapping and deadenylation of mRNAs in eukaryotes exposes them to what?
exonucleasees
95% of RNA is ______
noncoding RNA
miRNA/siRNA does what to translation
suppresses it
How does miRNA/siRNA suppress translation?
attaches to mRNA complementary
What happens if miRNA/siRNA attaches itself 100% complementary to mRNA?
degrades mRNA
What happens if miRNA/siRNA attaches itself less than 100% complementary to mRNA?
suppresses translation
________ strand of miRNA attaches itself to mRNA
leader strand
lncRNA is ___________ expressed
lowly
lncRNA forms a _____________ with DNA to recruit factors
triple helix
Does lncRNA usually affect transcription (nucleus) or translation?
transcription
circRNA sequesters __________
miRNA
cirRNA are resistant to _________
exonucleases
circRNA are produced by ________
back splicing
what are tRFs?
fragments of tRNAs
What do tRF regulate?
translation in stress conditions
What is the use for siRNA in research?
can downregulate genes
What is the use for miRNA in research?
mimics other miRNAs (tissue specific)
How does the SELEX process work?
finds RNA that will bind to a specific molecule
What is the advantage of the SELEX process?
can be used on live cells
What are the 4 advantages of RNA therapeutics?
safer
faster
personalized
tackle difficult targets
what are the 2 challenges of RNA therapeutics?
degrades quickly
delivery (charged and large)
Do prokaryotes have introns?
no
Regulation in multicellular organisms is important for cell _____________
specialization
What do master transcriptional regulators control?
cell identity
miRNA is transcribed by which RNA polymerase?
II
What is the most abundant RNA in a cell?
rRNA
What is RNAse P?
ribozyme
_____RNA is a signal recognition particle (SRP) in eukaryotes
7SL
What catalyzes the spliceosome?
U6 snRNA
What does U1 snRNA do?
binds to sequence complementary and aids spliceosome
In prokaryotes, what is the first thing to unwind?
-10 (pribnow) box
The -10 (pribnow) box is the first thing to unwind in prokaryotes. What is its sequence rich in to aid in winding?
AT
_________ act to turn off expression in both inducible and repressible operon
repressors
RPB-1 contain the ___________
CTD
______ phosphorylates Ser5-P for release of the mediator
TFIIH
What polymerase makes lncRNA?
II
________ phosphorylates CTD
TFIIH
Bacteria only need _____ transcription factor. What is it?
1
sigma factor
Lac repressor is a protein or lipid?
protein
Lactose controls _________
Glucose controls _______
Lac repressor
CAP
What charge do histone N terminal tails?
positive
Are histone N-terminal tails structured of unstructured?
unstructured
Where are histone modification most present on the histone?
N terminal tail
Can the charge of N-terminal tails of histones be changed?
yes
Do all lncRNA have 5’ caps?
yes
What is the function of snoRNAs?
base modification in rRNA and tRNAs
What RNAs are double stranded?
siRNA and miRNAs
tRNA transcripts are processed by…..
RNAse P, endonuclease, ligase
can methylated mRNAs be translated anymore?
yes
What is the catalytic part of the minor spliceosome?
U6atac
What is U11?
the non catalytic part of the minor spliceosome
What is 4.5SL RNA?
bacterial SRP
What is U2 snRNA?
non catalytic part of major spliceosome
