Exam 3 Flashcards
(263 cards)
1
Q
What are assays that determine RNA secondary structures?
A
RNAse Digestion
DMS-seq
SHAPE-seq
2
Q
What are the assays that determine RNA abundance?
A
(q) RT-PCR
PT-PCR
RNA-seq
3
Q
What is the assay that determines promoter/consensus sequences?
A
CHIP-seq
4
Q
What is the assay that determines rate of transcription?
A
GRO-seq
5
Q
What are the 3 enzymes used in RNAse digestion assay and what do they do?
A
RNAse V1: cleaves dsRNA
RNAse 1: cleaves ssRNA
RNAse T: cleaves ssRNA
6
Q
If you were using RNAse V1 to determine the secondary structure what would you see on the gel if the temperature was high?
A
nothing: RNAse V1cleaves dsRNA so it would all be denatured
7
Q
If you were using RNAse 1 to determine the secondary structure what would you see on the gel if the temperature was high?
A
everything: RNAse 1 cleaves ssRNA with would be all of the RNA at high temps
8
Q
What is the modification of DMS-seq?
A
modify A and C in ssRNA
9
Q
Is DMS-seq genome wide or sensitive?
A
genome wide
10
Q
What is special about DMS-seq?
A
it can be done in vivo and in vitro
11
Q
In DMS-seq where does the RT polymerase stop?
A
at all the modifications of the A and Cs
12
Q
Are there DMS signals present at dsRNA in DMS-seq charts?
A
no; it only deals with ssRNA
13
Q
DMS signals in DMS-seq represent what?
A
ssRNA
14
Q
What is the most accurate assay at predicting RNA secondary structures?
A
SHAPE-seq
15
Q
What is the modification of SHAPE-seq?
A
2’ OH on ssRNA
16
Q
What is special about SHAPE-seq that makes it so specific?
A
single nucleotide resolution (observes each base)
17
Q
SHAPE-seq and DMS-seq have the same mechanism but what 2 things are different about them?
A
- SHAPE-seq has single nucleotide resolution (more specific)
- different modifications
18
Q
What RNA abundance assays are relatively quantitative?
A
(q) RT-PCR
RNA-seq
19
Q
(q) RT-PCR and RT-PCR are the same except for what 2 things…
A
(q) RT-PCR is quantitative and measures every cycle while RT-PCR is not
20
Q
(q) RT-PCR and RT-PCR is genome wide or sensitive?
A
sensitive
21
Q
What RNA abundance assay uses fluorescence?
A
(q) RT-PCR
22
Q
What is the equation in (q)RT-PCR to determine the fold of a sample?
A
2 ^ (cycle # - cycle #)
23
Q
What RNA abundance assay is the most commonly used?
A
RNA-seq
24
Q
Is RNA-seq genome wide or sensitive?
A
genome-wide
25
What assay can detect intron splicing?
RNA-seq
26
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)
27
What are the dips in RNA-seq charts represent?
introns/splicing
28
Is CHIP-seq genome wide or sensitive?
genome-wide
29
What does CHIP-seq use to tag RNA?
antibodies
30
what does CHIP-seq idenitfy?
location of proteins that bind to DNA (like sigma factor)
31
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
32
CHIP-seq chart shows letters at positions in the genome; what does this help identify?
consensus sequence
33
What is the modification in GRO-seq?
changing UTP to BrUTP
34
What does GRO-seq use to tag BrUTP?
antibodies
35
What assays use antibody tagging?
GRO-seq and CHIP-seq
36
Is GRO-seq genome wide or sensitive?
sensitive
37
What assay looks at strands being actively transcribed by RNA polymerase?
GRO-seq
38
What assay needs different adaptors added to each end of fragments and why?
GRO-seq
needed to identify the direction of transcription
39
What does the first positive peak in GRO-seq graphs show?
proximal pausing
40
What 3 things did GRO-seq discover?
1. proximal pausing
2. bi-direction transcription
3. transcription (bi-directional) at enhancers even though they don't code for anything
41
What 3 things are special to RNA making it less stable?
1. 2' OH
2. single stranded
3. uracil (incorrect base pairing)
42
How many hydrogen bonds does G-C, A-U and G-U have?
G-C: 3
A-U: 2
G-U: 2
43
Out of G-C, G-U and A-U what is their strength ranking?
G-C (strongest)
A-U
G-U (weakest)
44
What is the most common unusual base in RNA?
pseudouridine
45
unusual bases are most common in what RNAs?
tRNAs
46
What is the purpose of unusual bases in RNA?
increase stability
47
What RNA assays are sensitive and not genome wide
(q)RT-PCR
GRO-seq
48
What does Non-Watson-Crick base pairing triple helixes do to RNA?
increase stability
49
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
50
What is different about A form RNA?
shorter and fatter
51
What is an example of a G-quadruplex?
GGGG (telomeres)
52
Are secondary or tertiary RNA structures more stable?
secondary (lower free E)
53
What proves structure = function in RNA?
ncRNA secondary structure are more conserved than their sequences
54
______________ are ribonucleoproteins which guide RNA molecules to their appropriate destinations
signal recognition particles (SRP)
55
eukaryotic SRPs are made of _____ RNA
7SL
56
What is pRNA?
packaging RNA
57
What are ribozymes?
catalytic RNA
58
What do natural ribozymes do?
formation of peptide bonds
cleave phosphodiester bonds
59
What do artificial ribozymes do?
glycosidic bond formation
RNA phosphorylation
60
What happens if RNA is a ribozyme AND a riboswitch?
1 sequence with 2 structures with different functions
61
What are riboswitches?
sensitive to environmental changes
62
What are aptamers?
RNA that binds to specific ligands
63
What is special about RNA in the RNA v. Lipid world that show they were first?
store genetic information and catalytic reactions
64
What were the first enzymes?
ribozymes
65
what RNA was the first that lead to life?
self-replicating RNA
66
What is transcription?
RNA synthesized from DNA template
67
what performs transcription polymerization?
RNA polymerase
68
What are 3 similarities between RNA and DNA polymerase?
require a template
goes in 5'--3'
adds single nucleotides at a time
69
Does RNA polymerase require a primer?
No (DNA polymerase does)
70
Can transcription happen outside of S phase in prokaryotes?
yes
71
Is RNA or DNA polymerase more error prone?
RNA polymerase
72
Are transcription and translation coupled in eukaryotes?
No (it is in prokaryotes)
73
How many RNA polymerases does bacteria have?
1
74
How many subunits does bacterial RNA polymerase have?
5
75
What is the prokaryotic holoenzyme of RNA polymerase?
the active form of RNA polymerase (sigma factor bound)
76
What is not always attached to prokaryotic RNA polymerase?
sigma factor
77
What does the prokaryotic RNA polymerase sigma factor do?
directs polymerase to promoter
78
Do all prokaryotes have the same number of sigma factors?
No all different
79
Does bacterial RNA polymerase have a helicase?
No, uses Mg2+ isomerase to unwind DNA
80
What are the 4 steps of bacterial transcription initation?
1. RNA pol sigma factor recognizes and binds to promoter (closed complex)
2. DNA winds by Mg2+ isomerazation (open complex) transcription bubble
3. first phosphodiester bond formed (unstable ternary complex)
4. release of sigma factor (stable ternary complex)
81
0
0
82
Do sigma factors have to bind to exact consensus sequences?
No, they can vary slightly
83
Why is it beneficial for bacteria to have different variations of consensus sequences?
allows different levels of that genes expression and different times
84
The more the consensus sequence matches the original...
the stronger the binding of the sigma factor = more transcription
85
0
0
86
What specifically on the promoter does the sigma factor recognize?
consensus sequence
87
The _____ domain on bacterial RNA polymerase binds the UP element, thereby strengthening RNA Pol’s binding affinity to the promoter
alpha - CTD
88
Upstream promoters are present on ________ expressed genes
highly expressed genes
89
What does DNA footprinting identify?
RNA polymerase binding site (sigma factor)
90
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
91
What is promoter escape?
in abortive initiation polymerase is able to escape and polymerize because sigma factor released
92
in bacteria, _________ forms ahead and behind transcription bubble during elongation
positive supercoils
93
In bacteria, is transcription a smooth process?
No (proximal pausing)
94
What stalls RNA polymerase in transcription in bacteria?
strong GC stem loop
95
In bacterial Intrinsic termination, what causes the RNA polymerase to fall off and release transcript?
UUUUUUU
96
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
97
Why is post-transcriptional modification in bacteria rare?
transcription and translation coupled
98
What is a post-transcriptional modification in bacteria?
polyadenylation
99
Polyadenylation in bacteria _______ the mRNA while in eukaryotes it _______ it
bacteria - destabilizes
eukaryotes stabilizes it
100
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
101
What degrades bacterial RNA?
endosomes and exosomes
102
Why is the polyA tail added to bacterial mRNA?
bacteria have hairpins at the end of mRNA so polyA allows exonucleases to degrade mRNA
103
What is constitutive gene expression?
expressed all the time
104
What is repressible gene expression?
normally off
105
What is inducible gene expression?
normally on
106
What regulation is most important in bacteria?
transcription regulation
107
What kind of system is Lac operon?
inducible -- negative and positive regulation
108
What Lac gene makes the repressor?
Lac I
109
What happens to cAMP levels when lactose is present?
high
110
What happens to CAP when lactose is present?
bound
111
What happens to RNA polymerase when lactose is present?
binds to promoter
112
What happens to Lac genes when lactose is present?
transcribed
113
What happens to Lac repressor when lactose is present?
allolactose blocks binding site so it doesn't bind to operator
114
What does Lac Z transcribe?
B-gala
115
What does Lac Y transcribe?
perm
116
What does Lac A transcribe?
transA
117
What happens to cAMP levels lactose is absent?
low
118
What happens to CAP when lactose is absent?
not bound
119
What happens to RNA polymerase when lactose is absent?
not bound
120
What happens to Lac genes when lactose is absent?
not transcribed
121
What happens to Lac repressor when lactose is absent?
binds to operator
122
Do does cAMP bind to CAP when lactose is present?
yes
123
Does cAMP bind to CAP when lactose is absent?
no
124
In the presence of glucose and no lactose look like what?
the same cycle as no lactose present
125
In the presence of glucose AND lactose, what happens to CAP?
not bound
126
In the presence of glucose AND lactose, what happens to the level of cAMP?
low
127
In the presence of glucose AND lactose, what happens to the repressor?
not bound
128
In the presence of glucose AND lactose, what happens to RNA polymerase?
weakly binds
129
In the presence of glucose AND lactose, what happens to the Lac genes?
little transcription
130
What type of system is Trp?
repressible system with 2 negative regulations
131
When Trp is high in the cell, what happens to the repressor?
Trp binds to repressor and they bind to operator
132
When Trp is high in cell, what happens to RNA polymerase?
not bound
133
What happens to Trp genes when Trp is high in cell?
no transcribed
134
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
135
When Trp is low in cell, what happens to RNA polymerase?
binds
136
What happens to Trp genes when Trp is low in cell?
transcribed
137
What is the second line of defense for Trp operon?
attenuation control
138
When Trp levels are high, what happens to the leader sequence?
hairpin loop is formed = no transcription
139
When Trp levels are high in a cell, what happens to the level of Trp-tRNA?
increase
140
When Trp levels are low in a cell does the polymerase stall at the leader sequence or pass through?
stall
141
What are riboswitches?
RNA that changes its secondary structure
142
Riboswitches are common in ___________
bacteria
143
sRNA bind ____________ to mRNA
complementary
144
one __RNA can regulate multiple target genes
sRNA (small RNA)
145
What sequence allows attenuation control of Trp operon?
leader sequence (can adopt 2 structures)
146
What does RNA polymerase I in eukaryotes transcribe?
rRNA
147
What does RNA polymerase II in eukaryotes transcribe?
mRNA
snoRNA
miRNA/siRNA
sRNA
lncRNA
148
What does RNA polymerase III in eukaryotes transcribe?
tRNA
sRNA
149
How many subunits does eukaryotic RNA polymerase II have?
12
150
How are eukaryotic II and prokaryotic RNA polymerases similar
structure
151
What assays use antibody tagging?
CHIP-seq
GRO-seq
152
What does transcription factor TFIID do in eukaryotic RNA polymerase II?
recognize TATA box (promoter)
153
What does transcription factor TFIIH do in eukaryotic RNA polymerase II?
unwinds DNA and releases polymerase from promoter (phosphorylation of CTD tail)
154
Is there a universal promoter sequence for eukaryotes?
no
155
Where are core promoters?
close to start site
156
Where are proximal promoters?
distant from start site
157
Where are distal promoters?
far from start site
158
How do eukaryotes fine tune levels of expression using promoters?
combinations of proximal and core promoters
159
How are proximal promoters used even though they far not near start site?
DNA folds and uses a mediator (loops form; neighborhood)
160
TFII___ binds to TATA box via the _____
TFIID
TBP
161
TFIIH has ______ activity and ________ CTD causing the release of mediator allowing polymerase to move
helicase
phosphorylation
162
What transcription factor in eukaryotes is important for promoter selection?
TFIID
163
TBP binding to promoter causes __________ in DNA making unwinding easier
bend
164
What are the 4 steps of eukaryotic transcription initation?
1. TFIID bind to TATA box
2. TFIIB is recruited to TATA box
3. RNA polymerase recruited with TFIIF
4. TFIIE and TFIIH join complex = complete pre-initiation complex
165
Eukaryotic transcription elongation requires...
elongation factors
166
eukaryotic RNA polymerase always does proximal ________
pausing
167
When does 7mGTP capping first happen in eukaryotes?
elongation
168
When does splicing start in eukaryotes?
elongation
169
What are the 3 ways nucleosomes are dealt with in eukaryotes?
1. chaperons displace nucleosomes
2. polymerase can move around them
3. co-transcriptional modification of histones can displace them
170
What are the co-transcriptional modifications in eukaryotes?
5' capping
splicing
polyadenylation
171
Explain the CTD cycle?
CTD contain a repeat with Serines that get phosphorylated and will recruit certain factors during transcription
172
What does Ser5-P do in transcription of eukaryotes?
recruits capping and splicing factors
173
What does Ser2-P do in transcription of eukaryotes?
recruits polyadenylation and termination factors
174
When is Ser5-P most abundant in eukaryotic transcription?
initiation/elongation because it recruits capping and splicing factors
175
When is Ser2-P most abundant in eukaryotic transcription?
elongation/termination because it recruits polyadenylation and termination factors
176
What facilitates co-transcriptional modifications in eukaryotic transcription?
CTD
177
What is the first transcription modification to be done in eukaryotes?
7-methylG capping
178
Are 5' caps added to all RNA polymerase II transcripts in eukaryotes?
yes
179
What kind of bond is made in 5' capping?
5'-5' phosphodiester
180
Why are 5'-5' phosphodiester bonds used in 5' capping?
resistant to exonucleases
181
________ group is added to the 2nd and 3rd nucleotides after the 5' cap
methyl group
182
5' capping aids in the first ________
intron splicing
183
Are polyA tails added to all RNA polymerase II transcripts?
no
184
Does intron splicing in eukaryotes have to be precise?
yes; it could cause a frameshift
185
are there more intron or exons?
introns
186
What mainly does the splicing on introns in eukaryotes?
spliceosomes
187
In major intron splicing in eukaryotes, what is recognized at the 5' end and 3' end?
5' -- GU (T)
3' -- AG
188
In minor intron splicing in eukaryotes, what is recognized at the 5' end and 3' end?
5' -- AU (T)
3' -- AC
189
Exon and intron boundaries are marked by ______
CTD
190
What are the steps of the spliceosome process?
1. A (branch point) is pushed out of sequence by snRNA
2. A pairs with G farther up the sequence (beginning of intron) forming a loop
3. loop is removed (intron)
4. splice site is marked by exon junction complex (EJC)
191
What is different about group II self-splicing compared to normal splicing?
same as regular but there is no spliceosome complex
192
What is different about group I self-splicing compared to normal splicing?
uses free G instead of A = no loop
no complex
193
Why do mammals have alternative splicing forms?
allows for diversity of the function of 1 mRNA (can be tissue specific)
194
What are the 3 options of transcription termination in eukaryotes?
1. allosteric model
2. torpedo model
3. combination
195
How does the transcription termination allosteric model work in eukaryotes?
RNA poly II is destabilized by the conformational change of sequence and falls off
196
How does the transcription termination torpedo model work in eukaryotes?
exonuclease degrades RNA from the 5' end and displaces RNA polymerase II
197
What does the post-transcriptional base-editing cause?
change in protein sequence
198
Base editing in eukaryotes in most common in what type of RNAs?
tRNAs
199
What is A to I base editing done by?
ADARs
200
A to I base editing is involved in ______ processing
miRNA
201
What is A to I base editing important for?
nervous system and development
202
What is C to U base editing done by?
APOBECs
203
mRNA methylation is the only modification that is ____________
transient (reversible)
204
in mRNA methylation, what are readers?
recognize and bind to modified bases
205
in mRNA methylation, what are writers?
modify bases
206
in mRNA methylation, what are erasers?
reverse modification
207
What are the 3 examples of mRNA quality control in eukaryotes?
1. non-stop decay
2. no-go decay
3. nonsense-mediated decay
208
When is the eukaryotic mRNA quality control non-stop decay recruited?
recruited when mRNA doesn't have a stop codon
209
When is the eukaryotic mRNA quality control no-go decay recruited?
when ribosome starts translation then stalls because an intron is not removed
210
When is the eukaryotic mRNA quality control nonsense-mediated decay recruited?
when stop codon is added before the real stop codon
211
Decapping and deadenylation of mRNAs in eukaryotes exposes them to what?
exonucleasees
212
95% of RNA is ______
noncoding RNA
213
miRNA/siRNA does what to translation
suppresses it
214
How does miRNA/siRNA suppress translation?
attaches to mRNA complementary
215
What happens if miRNA/siRNA attaches itself 100% complementary to mRNA?
degrades mRNA
216
What happens if miRNA/siRNA attaches itself less than 100% complementary to mRNA?
suppresses translation
217
________ strand of miRNA attaches itself to mRNA
leader strand
218
lncRNA is ___________ expressed
lowly
219
lncRNA forms a _____________ with DNA to recruit factors
triple helix
220
Does lncRNA usually affect transcription (nucleus) or translation?
transcription
221
circRNA sequesters __________
miRNA
222
cirRNA are resistant to _________
exonucleases
223
circRNA are produced by ________
back splicing
224
what are tRFs?
fragments of tRNAs
225
What do tRF regulate?
translation in stress conditions
226
What is the use for siRNA in research?
can downregulate genes
227
What is the use for miRNA in research?
mimics other miRNAs (tissue specific)
228
How does the SELEX process work?
finds RNA that will bind to a specific molecule
229
What is the advantage of the SELEX process?
can be used on live cells
230
What are the 4 advantages of RNA therapeutics?
safer
faster
personalized
tackle difficult targets
231
what are the 2 challenges of RNA therapeutics?
degrades quickly
delivery (charged and large)
232
Do prokaryotes have introns?
no
233
Regulation in multicellular organisms is important for cell _____________
specialization
234
What do master transcriptional regulators control?
cell identity
235
miRNA is transcribed by which RNA polymerase?
II
236
What is the most abundant RNA in a cell?
rRNA
237
What is RNAse P?
ribozyme
238
_____RNA is a signal recognition particle (SRP) in eukaryotes
7SL
239
What catalyzes the spliceosome?
U6 snRNA
240
What does U1 snRNA do?
binds to sequence complementary and aids spliceosome
241
In prokaryotes, what is the first thing to unwind?
-10 (pribnow) box
242
The -10 (pribnow) box is the first thing to unwind in prokaryotes. What is its sequence rich in to aid in winding?
AT
243
_________ act to turn off expression in both inducible and repressible operon
repressors
244
RPB-1 contain the ___________
CTD
245
______ phosphorylates Ser5-P for release of the mediator
TFIIH
246
What polymerase makes lncRNA?
II
247
________ phosphorylates CTD
TFIIH
248
Bacteria only need _____ transcription factor. What is it?
1
sigma factor
249
Lac repressor is a protein or lipid?
protein
250
Lactose controls _________
Glucose controls _______
Lac repressor
CAP
251
What charge do histone N terminal tails?
positive
252
Are histone N-terminal tails structured of unstructured?
unstructured
253
Where are histone modification most present on the histone?
N terminal tail
254
Can the charge of N-terminal tails of histones be changed?
yes
255
Do all lncRNA have 5' caps?
yes
256
What is the function of snoRNAs?
base modification in rRNA and tRNAs
257
What RNAs are double stranded?
siRNA and miRNAs
258
tRNA transcripts are processed by.....
RNAse P, endonuclease, ligase
259
can methylated mRNAs be translated anymore?
yes
260
What is the catalytic part of the minor spliceosome?
U6atac
261
What is U11?
the non catalytic part of the minor spliceosome
262
What is 4.5SL RNA?
bacterial SRP
263
What is U2 snRNA?
non catalytic part of major spliceosome
