MOLEC 2 Flashcards
1
Q
which techniques are used to detect RNA?
A
- FISH
- NB
- Microarrays
- assays
- Nuclear run on
- RT-PCR
- RNA seq
- Reporter gene
- hybridization
2
Q
which techniques are based on hybridization?
A
- NB
- Microarrays
- FISH
3
Q
how can u denature DNA?
A
heat or alkali solution
4
Q
how can u denature RNA?
A
heat or chemicals such as urea or formaldehyde
5
Q
which techniques allow you to determine the location of gene?
A
- FISH
6
Q
what does the stability of RNA depend on?
A
degradation rate
7
Q
which technique is best to compare between two samples?
A
microarrays
8
Q
which technique is best used to see the expression of gene under certain circumstances?
A
RNA-seq
9
Q
most expensive techniques?
A
microarrays and RNA-seq
10
Q
FC equation
A
log2 (exp/control)
11
Q
if FC =1 what does that mean?
A
expressed twice as much in the exp compared to control
12
Q
technique for quantifying signal intensities
A
densitomerty
13
Q
_______ intensity of each fluorescenet spot
____ number of reads associated with each gene
A
microarrays
RNA seq
14
Q
______ + _____ = GRO seq
A
RNA seq + Nuclear Run On
15
Q
limitation of reporter gene
A
decay rates influence this process
16
Q
techniques for measuring RNA length
A
run off transcription
G-less cassate
17
Q
in run off transcription why were TFs important?
A
they allow RNAP to recognize the right TSS, without it we will have RNAP starting at different locations and we get different bands
18
Q
techniques for detecting proteins
A
WB (immunoblot)
epitope tagging
19
Q
how to detect proteins in WB? hint: one word
A
chemiluminescene
20
Q
techniques that are used to examine protein protein interactions
A
IP and Co-IP
21
Q
techniques that are used to examine protein DNA interactions
A
EMSA filter binding assay
22
Q
in IP do the protein of interest, AB, and protein G/A go to the pallete or supernated section
A
pallete
23
Q
techniques for examining protein location in the genome
A
CHIP
24
Q
what is the starting material in CHIP
A
living cells - chromatin
25
which subunit within bacterial RNAP is important for enzyme assembly?
omega
26
consensus sequence and what does each letter represent?
WGNYC
W= A/T
G=guanine
N=any base
Y=pyrimidine (cytosine (C), thymine (T), and uracil (U))
C= cytosine
27
What element was found to be around 10 bp upstream to the TSS in bacteria, and was found to consist of 6-7 bp? select as many
a. -10 box
b. Pribnow box
c. TATA box
d. -35 box
-10 box
aka
Prinbow box
28
consensus sequences are on the
a. template strand
b. coding strand
coding strand
29
what elements make up the core promoter in E. coli?
-35 & -10
30
what element is found upstream to the core promoter? where is it mostly seen?
UP element, and seen in rrn genes
31
what is homology region 1? what about 2?3?4?
homology region 1: prevents the sigma from binding on its own to the DNA
homology region 2: 2.4 binds to the -10 box
homology region 3: binds the core RNAP well to the DNA
homology region 4: 4.2 binds the -35 box
32
what is required for the UP element to function?
the C-term of the ALPHA subunit
33
what forms the catalytic center of e.coli RNAP?
Two asp (D) within BETA PRIME bond to 1 OR 2 Mg2+
34
wrong bases are removed by ______ during bacterial elongation
ribonuclease (RNase)
35
what is each operon made up of?
1. multiple ORFs
2. terminator
3. promoter
4. regulatory regions
36
operon structure in order from 5'-3'
5'
Activator
Promoter
Operator
multiple ORFs
TES
3'
37
______ when there is a shift from one nutrient source to another
diauxric shift
38
lac operon structure in order from 5'-3'
5'
LacI
CAP site
Promoter
Operator
LAC Z
LAC Y
LAC A
TES
3'
39
what is meant by repression and de-repression in lac operon?
repression: when we have a repressor monomer produced by LACi that teterimizes and binds to the operator blocking the RNAP from forming lactose
de-rerpession: an inducer molecule formed from the isomerization of lactose into (1,6 linkage) known as allolactose or IPTG binds to the repressor tetramer changing its confirmation making an allosteric protein that also changes the confrimation of the operator and blocks the interacting between repressor and operator, therefore, lactose breakdown is achieved.
40
why are CAP-cAMP very weak?
because their -35 box does not match the consensus sequence well
41
with which component of RNAP do the a CAP interact with?
alpha CTD just like the UP element
42
______ CAP ______ interacts with _______ of ______ subunit of RNAP
one CAP monomer interacts with one monomer of alpha subunit of RNAP
43
structure of trp operon from 5' - 3'
5'
trpR
break
promoter
operator
trpL
attenuator
trpE
trpD
trpC
trpB
trpA
TES
3'
44
what forms the leader in trp operon?
trpL and attenuator
45
what forms the regulatory region in trp operon?
trpR
break
promoter
operator
trpL
attenuator
trpE
trpD
46
do co-activators or co-repressors bind to DNA directly or indirectly? give an example
they bind indirectly.
for example, trpR synthesizes aporepressor monomer that dimerizes and associated with CO-REPRESSOR trp that binds to it, and then the aporepressor component binds DIRECTLY to the operator (DNA element)
47
which regions of leader contain the intrinsic repeats?
3 and 4
48
which regions form within the leader when we have high Trp? what about low trp?
high: regions 3:4 and regions 1:2
low: regions 2:3
49
which regions form an anti-termination signal?
regions 2:3
50
explain the stalling of ribosome
ribosome stalling occurs at two codons in the trp region 1 because there isn't enough charged tRNA to bring and continue the translation process. This prevents region 1 from pairing with region 2, giving regions 2 and 3 the opportunity to form a hairpin resulting in an anti-termination signal, and trp synthesis can keep going.
51
sigma 70 is also _____
sigma 43 is also ____
sigma 70 is also sigma D
sigma 43 is also sigma E
52
stages of temporal control
aka in SPO1
first early phage genes
middle genes
late phage genes (NO MORE HOST GENES)
53
what carries the transcription of early phage genes in SPO1 infection?
host RNAP using sigma A
54
explain how early, middle and late genes transcribed.
Host core RNAP with sigma factor A produce early genes including the gp28 protein which is sigma specific.
gp28 with the host core RNAP will transcribe the middle genes that include gp33 and gp34
gp33 and gp34 contain THEIR RNAP which will transcribe the late genes
55
_______ is expressed during early sporulation
______ is expressed during enviromental stress
______ is expressed during high temp
_____ is expressed during N starvation
_____ is expressed during stationary phase
E is expressed during early sporulation
B is expressed during enviromental stress
H is expressed during high temp
N is expressed during N starvation
S is expressed during stationary phase
56
why is T7 unique? how is formed
has all factors required for transcription ie. promoter and its own RNAP.
once class 1 genes or early genes transcribed by host RNAP, a small gene encoded T7 is also transcribed, now T7 ALONE will carry the transcription of late genes (aka class 2-3)
57
what is meant by temperate phage? what are we referring to?
this refers to the lambda because it has two modes of infection which are lytic and lysogenic
58
when the lambda genome is integrated into the bacterial DNA, the host is now called ____ and the phage DNA is called____
when the lambda genome is integrated into the bacterial DNA, the host is now called LYSOGEN and the phage DNA is called PROPHAGE
59
what is the lambda repressor known as? what is its promoter? what does it block?
cI and its promoter is PRM. blocks PR and PL to allow only lysogeny
60
what does cI do?
binds to the operator and blocks the transcription else where in the lambda genome
61
what transcribes the genes in the lambda genome?
HOST HOLO-RNAP (WITH HOST SIGMA FACTOR)
62
during the lysogenic phage which gene is on?
ONLY cI
63
stages of lytic phase of lambda?
immediate early genes
delayed early genes
late genes
64
____ is similar to temporal control
lytic lambda mode
65
explain lytic phage in detail. mention the promoters and genes or any repressors if possible
the immediate early genes are transcribed by the host holoRNAP and terminate at intrinsic terminators
cro encodes the repressor of cI to ensure only the lytic phage is occuring (it blocks the PRM promoter to prevent lysogeny) and produce N
Then, if sufficient N or antiterminator factors are produced, the N will block the transcription of immediate early genes to allow the downstream transcription of delayed early genes from both sides (PL and PR), this is possible because the N binds to the but site on RNA transcript ensuring RNAP IGNORES any terminators and continues into the delayed early genes transcription
Then, once delayed genes are produced, factor Q is also produced which is another antiterminator factor
When Q is present it transcribes from the PR PRIME promoter to allow for FULL length transcriptions of LATE genes. This is possible because Q binds to the qut site on the DNA when the RNAP pauses shortly after delayed gene transcription allowing to keep transcribing late genes by releasing the pause complex
66
which of the following bind to the RNA transcript?
a. nut
b. rut
c. qut
d. all of the above
nut
rut
67
what triggers entery to lysogeny?
delayed early genes produce cII that allows for the production of cI that blocks the PR and PL promoters and allows lysogeny to occur
68
basic order of infection whether its lytic or lysogeny.
infection
immediate genes
delayed early genes
if lambda repressor (cI is produced from cII that was produced with delated early genes) then lysogeny
if NO lambda repressor then lytic (by first expressing cro)
69
what generates the first repressor after infection?
PRE
70
what is the decision between between lytic and lysogney?
once delayed early genes have been transcribed, the cII mRNA is also transcribed forming the cII protein.
if enough cII is present, the RNAP will bind to the PRE and allows for the cI production which blocks lytic phage from occurring.
if conditions favour lytic, then cII is degraded
71
what are two ways that allow lysogeny and not lytic pathway?
way 1: when the delayed late genes are produced a cII mRNA is also produced which is translated to the cII protein, if we have ACCUMULATION of cII then, the cII will recruit the RNAP to bind to PRE and allow for cI transcription, promoting the lysogenic phase
way 2: antisense and sense cro annealing. PR produced the sense cro, while the antisense cro is produced by the PRE. The antisense and sense cro sequences are complementary to one another, once they bind to each other they form a duplex that interefers with cro translation, therefore, allowing cI to be produced and lysogeny will be premited.
72
PRE produced the _____ orientation of cI and the ____ of cro
sense for cI
and
antisense for cro
73
immediate and delayed early genes are produced by _____ promoter/(s)
the delayed early genes are produced by ____ promoter/(s)
the cI gene are produced by the ____ promoter/(s)
immediate and delayed early genes are produced by PL and PR promoter/(s)
the delayed early genes are produced by PR' promoter/(s)
the cI gene are produced by the PRM and PRE promoter/(s)
74
what is one way to limit the lambda repressor?
we have OR1, OR2, OR3..
Repressor dimers (2 pairs) each binds to OR2 and the other binds to OR1.. OR1 and OR2 overlap with PR..
While OR3 overlaps with PRM.. SAME THING WITH LEFT SIDE.. all results in a LOOP
When the lambda repressor binds to OR3 it will block the cI production since OR3 overlaps with PRM.. FEEDBACK LOOP (preventing too much lambds repressor built up)
75
nutrient rich favours?
lytic phase
76
nutrient poor favours?
lysogenic phase
77
protease cleave ____ which exposes promoters _____ allowing for the ____ phase to occur in bacteria
protease cleaves repressors 1 and 2 bound to OR1 and OR2 which exposes promoters PL and PR allowing for the lytic phase to occur in bacteria
78
what is unique about the N term in repressor in bacteria?
it has the helix-turn-helix motif (DNA binding domain)
79
in helix turn helix motif, the ____ is the recognition helix and the ___ is the motif
in helix turn helix motif, the third is the recognition helix and the second is the motif
80
where does each of the eukaryotic RNAPs function?
RNAP1 in the nucleolus
RNAP2/3 in the nucleoplasm
81
what is transcribed by RNAP2?
mRNAs
most snRNAs
miRNAs
82
what is transcribed by RNAP1?
most of the rRNAs
83
what is transcribed by RNAP3?
tRNAs
5S rRNAs
U6 snRNAs
other small RNA
7SK
84
how to convert from mg/mL to microgram/mL?
how to convert from g/mL to microgram/mL?
how to convert from ng/mL to microgram/mL?
how to convert from mg/mL to microgram/mL?
- times by 1000
how to convert from g/mL to microgram/mL?
- time by 1000000
how to convert from ng/mL to microgram/mL?
- divide by 1000
85
how many subunits does each RNAP contain?
RNAP1 14
RNAP2 12
RNAP3 17
86
which aa have S?
C
M
87
which aa have P?
NONE!
88
what is the largest subunit in RNAP2?
Rpb1
89
match:
gene
yeast protein
protein
human protein
mutation
RPB1
Rpb1
RPB1 (italics)
rpb1
RPB1- human protein
Rpb1- yeast protein
RPB1 (italics) - gene
rbp1 - mutation
90
ortholog to the following subunits:
Rpb1
Rpb2
Rpb3
Rpb1: Beta prime
Rpb2 : beta
Rpb3: alpha
91
what are the subunits that are shared among all eukaryotic RNAPs?
Rpb5
Rpb6
Rpb8
Rpb10
Rpb12
92
throughout the course we mentioned CTD a lot, which components rely on CTD?
1. CTD of RNAP alpha subunit interacts with UP element
2. one monomer of CAP interacts with one monomer of alpha subunit of RNAP
3. important for capping
4. guanylyl transferase binds directly to ser5 on ctd
5. Rpb1 contains the CTD which gets p
6. splicing factors coordinated by CTD
7. RNAP2 CTD promotes cleavage of pre-mRNA during polyA
93
what is A? name and property
Ala
non-polar
94
what is R? name and property
Arg
basic
95
what is K? name and property
lys
basic
96
what is H? name and property
His
basic
97
what is D? name and property
asp
acidic
98
what is E? name and property
Glu
acidic
99
what is G? name and property
Gly
non-polar
100
what is P? name and property
Pro
non-polar
101
what is M? name and property
Met
non-polar
102
what is I? name and property
Ile
non-polar
103
what is L? name and property
Leu
non-polar
104
what is V? name and property
Val
non-polar
105
what is S? name and property
Ser
polar
106
what is F? name and property
Phe
aromatic
107
what is W? name and property
Trp
aromatic
108
what is Y? name and property
Tyr
aromatic
109
what is T? name and property
Thr
polar
110
what is N? name and property
Asn
polar
111
what is C? name and property
Cys
polar
112
what is Q? name and property
Gln
polar
113
which amino acids get phosphorylated in eukaryotes and prokaryotes?
Euk:
S
T
Y
prok:
His
114
which amino acids get mono/di/tri-methylated?
K: mono/di/tri
R: mono/sym di/asym di
115
which amino acids get acetylated?
SPECIFIC K
116
which amino acids get Ubiquintated?
K
117
Match:
TATA box containing
TATA box less
house keeping genes
consitituve expression
facultative expression
inducible genes
Development
Tissue Specific
TATA box containing:
facultative expression
inducible genes
Tissue Specific
TATA box less:
house keeping genes
consitituve expression
Development
118
proximal promoters are position ______ and orientation _______
enhancers are position ______ and oritentation______
proximal promoters are position DEPENDENT and orientation INDEPENDENT
enhancers are position INDEPENDENT and oritentation INDEPENDENT
119
what are the important elements in 5S rRNA and tRNA? where are they found?
5s rRNA: BOX A/C and downstream to TSS
tRNA: BOXA/B and downstream to TSS
120
what are non-classical promoters?
Examples:
1. U6 snRNA: transcribed by RNAP3 and has elements such as DSE-PSE-TATA upstream to TSS
2. U1/U2 snRNAs: transcribed by RNAP2 and have elements such as DSE-PSE upstream to TSS
121
proximal promoter in Euk but ______ in yeast
UAS
122
which of the following are DBP?
a. enhancers
b. silencers
c. activators
d. SSTFs
e. Co-activators
f. mediators
g. trp
h. aporepressor
i. inducer
j. allolactose
k. lac repressor
l. GTFs
c. activators
d. SSTFs
h. aporepressor
k. lac repressor
l. GTFs
123
which elements are needed for TAFs to bind to and increase Transcription level? which specific TAFs bind to each element?
INR : TAF1/2
DPE: TAF6/9
124
which element stabilizes the DNA-TBP binding?
TFIIA
125
which of the following are heterodimers?
a. TFIIA
b. TFIIB
c. TFIIE
d. TFIIH
e. TFIIF
A
E
F
126
which elements are required for TFIIB?where are they found?
BRE
TBP
upstream to TSS
127
mediator can be considered a
coactivator or GTFs
128
which of the following are negative elongation factors?
a. p-DSIF
b. p-NELF
c. DSIF
d. NELF
e. pTEFb
c. DSIF
d. NELF
129
importance of pausing?
posiing of RNAP for rapid gene activation
coordinating pre-mRNA processing
permissive chromatin
allowing another oppurtinuity for gene regulation
130
which motifs contain Zn?
zif268
Gal4
131
which motifs dimerize?
a. zif268
b. gal4
c. homeodomains
d. Gcn4
e. MyoD
gal4
gcn4
myoD
132
enhanceosome vs enhancer complex
enhancer complex: the activators are independent
enhancesoome: activators are dependent (need all)
133
Gcn4 vs Gcn5
Gcn 4: belongs to the bZIP motif
Gcn: HAT (coactivator)
134
enhanceosome steps
enhanceosome complexes bind to the enhancer all together --> recruit Gcn5 to bind to them --> Gcn adds Ac to the K --> recruitment of kinase component --> recruitment of SWI/SNF that shift the nucelosome away and expose TATA box --> TFIID interacts with TATA and Ac K via TAF1
135
functions of introns
- enhance gene expresson
- splicing slows down expresson
- alternative spilicing increase diversity
- regulatory RNAs seen in some introns
136
where does splicing take place?
ENTIRELY in the nucleus
137
list the splicing signals in order from 5' to 3'
5'
5'SS
BP
PT
3'SS
3'
138
rxn of splicing
5'GpGU_____A(OH)______AGp3'
OH attacks p in GPGU forming the 5'G-OH and
GUPA_______AGp3'
the the OH attacks the p on AGp3' forming the E1-E2 and lariate which is A____AG__OH
139
which of the following are ribnucleoprotein?
a. spliceosome
b. ribosomes
c. snRNA
d. hnRNA
all
140
splicesome snRNAs assembly
U1 associates with 5'ss
U2 associates with the sequences around BP including the A in BP
U4 interacts with U6 (to block activity), and U6 binds to U1
U5 binds to the exonic part of 5'ss
U2AF recognizes the 3'ss (65,35)
U1 dissociates
U4/U6 interaction is disrupted
U6 replaces U1 at 5'ss
U2 remains associated with BP-A
forming the U6-ISL loop and first step is catalyzed
141
list capping enzymes and what they do briefly
1. RNA triphosphatase cleaves the y phopsphate
2. guanylyl transferase (GMP attack to beta p from mRNA) = 5'-5' triphosphate linker
3. RNA (G-N7) methyltransferase - adds ch3 to n7 of G
142
what adds CH3 to 2'OH
2'O-RNA methytransferase
143
speaking of RT-PCR, if we see one really dark band on the bottom and one very light band on top what could this mean?
dark band at the bottom means that only consistuve exons are expressed since the ALT exon sites are weak and this is why the top band is light
144
speaking of RT-PCR, if we see one really dark band on the top and one very light band on bottom what could this mean?
dark band on top means the SR is bound to ALT exon (ESE) and allowed for ALT to be included while light band on bottom means the consitituve exons only
145
speaking of RT-PCR, if we see one really dark band on the bottom and ALMOST NO band on top what could this mean?
dark band on the bottom means that only consistive exons, and almost no band on top means that we have hnRNPs bound to ALT ensuing it is NOT included at all
146
which polymerase transcribes the pA or PAS?
2
147
structure of pA signal
USE (20nt)AAUAAA(10-30nt)CA(0-40 break) DSE
148
in order list the proteins involved in cleavage and polyadynelation
5-m7G:
CPSF associated with AAUAAA, the 73 unit is endonuclease that cleaves the transcript
PAP- which is a POLYA polymerase that adds A res to the 3'OH
CF1/CF2 which are cleavage factors
RNAP2 CTD promotes cleavage of pre-mRNA
CstF binds to DSE element
149
steps of cleavage according to torpedo
CPSF binds to the AAUAAA polyadenylation signal on the pre-mRNA.
CstF and CFs help cleave the transcript downstream of AAUAAA.
The 73 kDa subunit you mentioned is CPSF73, an endonuclease that cleaves the pre-mRNA.
This exposes a 3' OH end, to which PAP (poly(A) polymerase) adds adenine residues = poly(A) tail.
After cleavage, RNA pol II keeps transcribing → this leaves an uncapped 5' end on the leftover RNA.
Xrn2 (a 5'→3' exonuclease, also called Rat1 in yeast) binds the exposed 5' end.
Xrn2 degrades the RNA toward RNA pol II.
When Xrn2 catches up to the polymerase, it disrupts transcription, causing termination
150
techniques for examining how RNAs are processed in vivo
RT-PCR
RNase protection assay
RNA-seq
151
which one is first distal or proximal?
proximal then distal
proximal = short product
distal = longer product
152
increased CPSF and CstF leads to ______ promoter usage and therefore, a _____ product
hint: cancer cells
proximal
short
153
which is stronger distal or proximal?
distal
154
ser5 is mostly abondant at ______ while ser2 is mostly abundant at _____
ser5 is mostly abondant at START while ser2 is mostly abundant at END
155
kinases for Ser2 P at 3'
BUR1
ctk1
156
what are they two highly student Histone modifcations?
H3K4
H3K36
157
can H3K4 methylated?
yes, mono di, or tri
158
can H3K36 methylated
yes tri only!
159
H3K4 is associated with ____ end whule H3K36 with the _____ end
H3K4 is associated with 5' end whule H3K36 with the 3' end
160
______ histone methylatransfers for H3K4 and ______ for H3K36
SET1 histone methylatransfers for H3K4 and SET2 for H3K36
