1
Q
T/F the central dogma can be transferred back from protein to either protein or nucleic acid
A
false
2
Q
what can trigger reverse transcription
A
single strand RNA viruses (retroviruses)
3
Q
what is an example of a ssRNA virus that is involved with RNA replication
A
SARS-Cov-2 (Covid-19)
4
Q
what does a ssRNA virus replicated lead to
A
making of prions
5
Q
what is involved in the making of a nucleotide
A
- nitrogen base
- carbon sugar (deoxyribose)
- phosphate group
6
Q
How is DNA directional in that the two ends are different
A
- phosphoryl group = 5’ carbon of sugar
- free hydroxyl = 3’ carbon of sugar
- written 5’ to 3’ directionality
7
Q
how is the double helix stabilized
A
- hydrogen bonds and hydrophobic stacking
- edges of base pairs make hydrogen bonds
- base stacking is caused by the hydrophobic effect and van der Waals
8
Q
what way does the double helix wind up
A
right-handed (10-10.5 base pairs per turn)
9
Q
adenine forms _____ hydrogen bonds with thymine, while guanine forms ______ hydrogen bonds with cytosine
A
2; 3
10
Q
Chargaff’s rules
A
the amount of adenine equals the amount of thymine, the amount of guanine equals the amount of cytosine
purines = pyrimidines
11
Q
phosphodiester bond
A
- between the 3’ hydroxyl of sugar group in a nucleotide and phosphate group of 5’ carbon of another sugar group
12
Q
glycosidic bond
A
- nitrogen-carbon linkage
- 9’ nitrogen of purine bases or 1’ nitrogen of pyrimidine bases and the 1’ carbon of sugar group
13
Q
what is the melting temperature (Tm)
A
temperature at which half of the DNA strands are in the random coil or single-stranded (ssDNA) state
14
Q
what are the major and minor grooves in DNA
A
presence of grooves allows access to the hydrogen-bonding capabilities to exposed bases
15
Q
what are the major and minor grooves in DNA lined by
A
sequence-specific hydrogen bonding groups
16
Q
what does the hydrogen bonding capability in the major and minor grooves of DNA provide
A
means of sequence-specific interactions between DNA and the molecules it must interact with during replication and transcription
17
Q
what amino acids make up the largest number of hydrogen bonds
A
arginine
lysine
threonine
asparagine
glutamine
18
Q
T/F for A, C, T, G, the minor groove site is always near the glycosidic bond side
A
true
19
Q
DNA in what form has a major and minor groove
A
B form
20
Q
intercalating agents
A
hydrophobic molecules containing flat aromatic and fused heterocyclic rings insert between stacked base pairs of DNA
21
Q
T/F intercalating agents are potential cancer-reducing agents
A
true (either kill cancer cells or induce mutations)
22
Q
what are examples of intercalating agents and where are they most present
A
ethidium bromide
acridine orange
actinomycin D
mostly in minor grooves of DNA
23
Q
what are the full steps in prokaryotic DNA synthesis
A
1- separation of two complementary DNA strands
2- formation of replication fork
3- priming the replication
4- direction of DNA replication
5- elongation of replication chain
6- excision of RNA primers and their replacement by DNA
7- ligation of the nick DNA
24
Q
where does replication start in prokaryotic DNA synthesis (where to open double helix)
A
origin of replication - AT-rich, recognized by DnaA
25
replication is ____directional
replication is ______conservative
bi; semi
26
what does semi-conservative mean
one strand is from the parent, and the other is newly synthesized
27
how is the replication fork formed (how to open the double helix)
DNA helicase: bind to ssDNA and unwind the DNA double helix, ATP (break H bonds between strands)
28
after DNA helicase unwinds the double helix, how does it stay unwound
single strand binding protein (SSB)
- bind to ssDNA
- keep ssDNA separated
- protect DNA from cleavage
(break phosphodiester bonds)
29
how do you prime for replication in prokaryotic DNA synthesis (how to start DNA replication)
DNA polymerase adds nucleotides to an existing strand (needs a primer, cannot act on de-novo pathway because it cannot start from scratch)
- 3' OH group (phosphodiester bond formation) adding nucleotides to this end
- DNA polymerase only synthesizes DNA from 5' to 3'
30
how do you elongate the leading and lagging strands (how to extend the polynucleotide chain)
leading - new DNA strand, synthesized in the direction of replication form (5' to 3') replicated continuously
lagging - DNA strand at the opposite side of replication fork (3' to 5') replicated discontinuously
31
Okazaki fragments
short strand of DNA that were replicated discontinuously on the lagging strand
32
how to removes the RNA primer and fill in the gaps of the lagging strand
DNA poly III - elongates RNA primer until another stretch of RNA is encountered
DNA poly I - excises RNA primer, one nucleotide at a time
DNA poly I - fill gap
DNA-ligase - nick is sealed
33
DNA polymerase I has two jobs, what are they
removes RNA primers (5' to 3' exonuclease), and fill the gap
34
what two functions does DNA polymerase III have
- 5' to 3' polymerase activity, DNA synthesis
- 3' to 5' exonuclease activity, proofreading (will remove mismatched pair)
35
how to get rid of supercoils in prokaryotic DNA synthesis
DNA topoisomerases - bond covalently to the DNA phosphate as they break the phosphodiester linkage between neighboring nucleotides, storing the energy in that bond to use in releasing
36
DNA topoisomerase I
single strand break in double helix
- will cut one strand to rid of supercoil
- tighter supercoil
37
DNA topoisomerase II
double strand break in DNA
- will cut both strands
- end is relaxed circle
38
antimicrobials of topoisomerase II (drugs that inhibit supercoil end and will allow coils to form and stop replicating)
ciprofloxacin - DNA gyrase
quinolones
39
5' to 3' exonuclease, remove primer
3' to 5' exonuclease proofreading
specifically lagging strand
DNA poly I
40
3' to 5' exonuclease, proofreading
DNA poly III
41
differences between eukaryotic and prokaryotic DNA replication
- multiple origins versus single origin
- S phase in cell cycle (DNA replication)
- multiple polymerases
- multiple enzymes
42
Rnase H
enzyme in eukaryotic DNA replication
- removes RNA primers
43
what are the two enzymes in eukaryotic DNA replication that differ from prokaryotic DNA replication
Rnase H
topoisomerase
44
Anti-cancer drugs that stop supercoil replication
etoposide- human topoisomerase II
camptothecin- topoisomerase I
indenosioquinoline- topoisomerase I
45
how is eukaryotic DNA found
packaged with protein, forming a substance called chromatin
- Do not normally exist as the double helix
46
what is the structure of the eukaryotic nucleosome and how many histones are present
8 histones of the core particle - arranged as octamer (H2A, H2B, H3, H4)
- DNA wraps around the outside of the octamer
- beads on a string
47
what are the steps for the compaction of DNA into a eukaryotic chromosome
1- naked DNA
2- nucleosome beads (histones+DNA)
3- nucleofilament (30nm fiber)
4- nucleofilament is coiled and anchored to scaffold proteins
48
how do nucleosomes arrange themselves
- 30-nm fibers
- two interwound left-handed helical stacks
- linker DNA crossing the interior of the fiber
- folding generates chromosomes
49
how to replicate the end of the DNA of the lagging strand?
1- removal of RNA primer leads to the sorting of the chromosome after each round of replication
2- chromosome sorting eventually leads to cell death (cannot be synthesized as they get shorter and shorter - eukaryotic)
50
telomerase
eukaryotic
- replicated the ends of linear DNA molecules
- enzyme made of protein and RNA subunits
- elongates chromosomes by adding TTAGGG (telomeric DNA) to end of existing chromosomes
51
telomeric DNA sequence
TTAGGG
52
an RNA sequence in telomerase acts as a template for ____. This enzyme adds the telomeric sequence to the 3' end of the chromosome by its ______ _________ activity
DNA; reverse transcriptase
53
T/F high telomerase activity in normal somatic cells
false
54
without telomerase, what happens
the chromosomes are shortened each time the cell divides
55
telomerase activity in cancer
- high activity
- increased by 80-90%
56
what are the 3 activities of reverse transcriptase in DNA synthesis
- RNA-dependent DNA polymerase (5' to 3')
- ribonuclease H (5' to 3' exonuclease)
- DNA-dependent DNA polymerase (5' to 3')
57
a high affinity (100 fold higher) of r4everse transcriptase leads to what
a lack of repair system to remove it
58
nucleoside analog
modification on the hydroxyl group at the 3' carbon of the deoxyribose
59
vidarabine
inhibitor of DNA synthesis
- Ara A
- antiviral (herpes complex)
60
cyarabine
inhibitor of DNA synthesis
- Ara C
- anticancer (leukemia)
61
what is the target of chemotherapy, and what drugs help combat this
DNA replication; methotrexate -> dihydrofolate reductase and fDUMP (5-fluoracil) -> thymidylate synthase
62
cisplatin
chemotherapeutic agent that reacts with DNA:
- nitrogen's of adjacent purines (guanine) replace with chloride atoms
- modification disrupts DNA structure by cross linking two purines and leads to cell death
63
examples of highly proliferative cells
bone marrow
gut epithelium
skin/hair follicles
(no G0, very short G1)
side effects of chemotherapy
64
what gene codes for breast cancer
TP53
65
what gene codes for colorectal cancer
KRAS
66
examples of endogenous DNA damage
- replication errors (mismatches)
- spontaneous loss of nucleotides
- chemical exposure
(base alteration)
67
examples of exogenous DNA damage
- chemical exposure (base alteration)
- UV light exposure (thymine dimer)
- ionizing radiation (double strand break)
68
what is the estimate that each cell undergoes DNA damaging events per day
over 20,000
69
chromosomal mutations
involve changes to the entire chromosome or sections of it
- also called segmental mutations
70
chronic myeloid leukemia is what kind of mutation
chromosomal
71
point mutations
changes to one or a few base-pairs in the DNA in the form of substitution, deletion, insertion
72
point mutation - substitution
transitions and tranversions
through DNA replication
transition - purines (A->G) or pyrimidines (C->T through methylation/deamination)
transversion - purine -> pyrimidine and vice versa
73
mismatch point mutation (proofreading)
errors is DNA replication
proofreading - 1 in 1 mil bp
wo proofreading - 1 in 1000 bp
(cannot completely avoid)
74
D400A mutation in mice (DNA proofreading)
deaths of the mutant homozygotes were all due to malignancies
- proofreading activity is inhibited, cannot fix pairs and lead to death
75
oxidative deamination base alteration point mutation
- spontaneous (NH2->O)
- G:C pair to A:T pair mutation (transition)
76
A;T->G:C transition oxidative deamination base alteration
adenine -> (adenine deaminase) -> hypoxanthine
77
C:G->T:A transition oxidative deamination base alteration
5-methylcytosine -> (cytosine deaminase) -> thymine
- most common point mutation in cancer cells
78
oxidation: ROS such as hydroxyl radical base alteration point mutation
guanine -> (oxidized) -> 8-oxoguanine
- guanine is the base component in dGMP in DNA, can be oxidized to 8-oxoguanine
- 8-oxoguanine can pair with adenosine, not cytidine
G:C->T:A transversion
ROS induces DA damage
79
what is the most oxidative lesion observed in duplex DNA
8-oxoguanine
80
alkylation base alteration point mutation
chemical reaction that entails transfer of an alkyl group
- G:C->T:A transversion
- cytochrome P450 forms a free radical; a reaction with DNA forms an alkylated guanine
81
what is the most common type of alkylation
methylation
82
depurination base alteration point mutation
cleavage of the glycosidic bond connecting to the backbone
- destabilize the covalent bond to deoxyribose forms (AP sites)
- misread by the DNA polymerase
83
T/F the deamination, oxidation, methylation and depurination, which together may alter thousands of bases per cell genome each day, greatly exceeds the amount of damage created by exogenous mutagenic agent in most tissues
true
84
thymine dimer
UV radiation energy leads to cross linking of adjacent pyrimidines (thymine) along one strand of DNA disrupting the normal base pairing
85
double strand break
ionizing radiations (free radical products) of oxidative metabolism
- radiation can break ester bond and create the double strand break
86
what are typical cell responses to DNA damage
- cell cycle check point activation
- damage tolerance
- DNA repair
- apoptosis
87
cell cycle check point activation
pauses the cell cycle and gives the cell time to repair the damage before continuing to divide
G1 checkpoint - G1 arrest (prepare correct cells for replication)
G2 checkpoint - G2 arrest (prepare cells for division)
88
pRb and cell cycle machinery
Go/early G1 - pRb physically associates wit E2F factors and BLOCKS transactivation domain
late G1 - pRb releases E2F, allowing the expression of genes that ENCODE PRODUCTS necessary for S-phase progression
89
cyclin D1
product of bcl-1 gene
90
pRb stands for
phosphorylates retinoblastoma protein
91
what does p21 transcription factor target
p53 -> p21 is an inhibitor of S phase when there is DNA damage present
92
radio and chemotherapy cause what kind of DNA damage and what is the repair system
double-strand break
DSB repair
93
UV light causes what kind of DNA damage and what is the repair system
helix-distorting damage
nucleotide excision repair
94
replication errors cause what kind of DNA damage and what is the repair system
mismatches/insertions
mismatch repair
95
alkylating agents cause what kind of DNA damage and what is the repair system
O6-alkyl-guanine
direct reversal
96
reactive-oxygen species cause what kind of DNA damage and what is the repair system
single-strand breaks
singe-strand break repair
97
base damage to DNA leads to what repair mechanism
base excision repair
98
direct reversal repair
NO DNA REPLICATION
- do not require a template (can act de-novo)
- reversal mechanisms are specific to the type of damage incurred and do not involve breakage of the phosphodiester bond
99
methylation of guanine bases is directly reversed b
the protein methyl guanine methyl transferase (MGMT)
100
AP endonuclease is involved in what DNA repair mechanism
base excision repair
101
thymine dimer is involved in what DNA repair mechanism
nucleotides excision repair
102
base excision repair
- in the repair of damaged DNA (8-oxo-deoxyguanine, 3-methyladenine, deaminated bases)
- corrects DNA damage from oxidation, deamination, and alkylation
103
what enzymes are used in base excision repair
uracil DNA glycosidase (glycosylase)
AP endonuclease
DNA poly I and DNA ligase
104
nucleotide excision repair steps
1- identification of mismatched/mutated strand
2- nick by endonuclease, remove by exonuclease to create a gap
3- 5'->3' DNA poly fill gap
4- DNA ligase forms phosphodiester linkage
105
UV-induced thymine dimer repair steps
1- identification of mismatched/mutated strand
2- excision endonuclease (cut out and remove) also called excinuclease or UV specific endonuclease
3- 5'->3' DNA poly fill gap
4- DNA ligase forms phosphodiester linkage
106
double strand break repair
(relies on DNA replication)
- non-homologous end joining (NHEJ)
- homologous recombination repair
107
inherited mutations that affect DNA repair genes are strongly associated with high _______ risk in humans
cancer
108
hereditary nonpolyposis colorectal cancer (HNPCC)
mutations in the DNA mismatch repair pathway
109
xeroderma pigmentosum (XP) cancer
at least 8 distinctive genes
- 7 involved in NER
(defect to make thymine dimer)
110
ataxia telangiectasia (AT) cancer
small number of cancer cases
111
almost ____% of all identified familial _____ cancers involve germline transmission of a mutant BRCA1 or BRCA2 allele
50%; breast
112
___ to ___% of all familial ______ cancers are due to germline alleles of BRCA1 or BRCA2
70 to 80%; ovarian
113
xeroderma pigmentosum (XP)
- autosomal recessive disease due to deficiency of excinuclease
- first disease caused by defective DNA repair
- multiple basal cell carcinomas/skin malignancies at young age
114
carcinogens vs procarcinogens
carcinogens - agents that cause cancer DIRECTLY (radiation, chemicals)
procarcinogens - precursor of carcinogen (activated in metabolism)
115
___% of cancer cases caused by environmental and food carcinogens
80%
116
T/F there is a low correlation between mutagenesis and carcinogenesis
false, high correlation
117
carcinogen -> damaged DNA
procarcinogen (hydrophobic) -> liver processing (hydrophilic) -> forms BPDE (covalent high energy bond to guanine) -> DNA damage
118
what is the ultimate carcinogen
BPDE
119
what provides cytochrome P450
rat liver lysates (monooxygenases)
120
conversion of procarcinogens to carcinogens by cytochrome P450
procarcinogen -> cytochrome P450 -> alkylating agents (covalent high energy bond to guanine) -> pairs with A and not C, mutation present -> Covalently bound to DNA to form DNA adduct
121
vinyl chloride and styrene
procarcinogens
122
T/F intercalating agents are different from alkylating reagents
true, intercalating agents have a non-covalent interaction
123
Ames test-assays for carcinogenicity
- rat liver lysates to provide cytochrome P450
- Salmonella his- strain: must be grown in presence of histidine, and mutant allele is susceptible to BACK-MUTATION to wild-type
- salmonella cannot grow in histidine without operon -> lead to a mutation
- END OF TEST -> count bacterial colonies that underwent mutation allowing them to grow without added histidine
124
why is the small amount of rat liver added to the Ames test
to mimic the mammalian metabolism
125
oncogenes
- cancer causing agents
- proto-oncogenes are corresponding normal cell genes responsible for normal cell growth/division
- conversion of a proto-oncogene to an oncogene can lead to normal stimulation of the cell (gain of functions)
126
proto-oncogene to normal growth-stimulating protein through translocation/transposition
gene moved to new locus, under new controls
127
proto-oncogene to normal growth-stimulating protein through gene amplification
multiple copies of the gene
128
proto-oncogene to normal growth-stimulating protein through point mutation
within a control element
129
proto-oncogene to hyperactive or degradation resistant protein (cancer cell feature)
within the gene
130
tumor-suppressing genes
(opposing oncogenes)
- help prevent uncontrolled cell growth
- mutations that decrease protein products of tumor-suppressing genes may contribute to cancer (loss of function, defect on two copies)
- proteins: repair damaged DNA, inhibit the cell cycle in cell-signaling pathway
131
mutations in the Ras gene can lead to production of a _____________ Ras protein and increased cell division
hyperactive
132
Ras: oncogene
GTP-bound state is the "on" state
GDP-bound state is the "off" state
133
p53
tumor suppressor
134
multistep model of cancer
1- loss of tumor suppressor gene APC
2- activation of Ras oncogene
3- loss of tumor suppressor gene DCC
4- loss of tumor-suppressor gene p53
135
T/F RNA chains can be initiated de novo
true, they do not need a primer
136
antisense and sense strand of RNA
antisense - complementary to DNA template
sense - coding, identical to DNA nontemplate
137
T/F RNA polymerases are highly selective
true
138
transfer RNA (tRNA)
15%
- one specific type of tRNA for each of the 20 AA
- adaptors between AA and the codons in mRNA
139
ribosome RNA (rRNA)
80%
- structural and catalytic components of ribosomes
- facilitate the binding and positioning of the mRNA on the ribosomes
140
messenger RNA (mRNA)
5%
- carry genetic information from DNA to cytosol
- template for protein synthesis
141
what are examples of other non-coding RNAs
snRNA, miRNA, IncRNA
142
what are the following requirements for RNA polymerase (transcription)
1- template: newly synthesized RNA is complementary to the DNA template
2- activated precursors: form of 4 ribonucleotide triphosphates
3- divalent metal ions: Mg2+ or Mn2+
143
coding strand
DNA strand that has the same sequence as the RNA product
144
holoenzyme
sigma region
alpha region
Beta region
(sigma+core enzyme)
sigma - recognize promoter regions (initiation)
alpha/beta - core enzyme RNA polymerase activity (elongation)
Beta - termination
145
3 steps in prokaryotic RNA synthesis
initiation
elongation
termination
146
initiation
recognition of a specific DNA sequence by RNA polymerase and the beginning of the bond formation (promoter regions)
147
elongation
RNA polymerase continues the binding, bond formation, and translocation cycle
- unwinding of the 17 bases after binding of polymerase
- no primer required
- has helicase activity (prokaryotic)
- use ribonuclease triphosphate, release pyrophosphate
- DNA topoisomerases I and II relax supercoils
148
termination
the ends of genes are recognized by the RNA polymerase complex (B-dependent and independent)
- RNA poly stops moving on DNA template and the transcript falls off from the transcription complex
149
promoters
specific DNA sequences that direct RNA polymerase to the proper initiation site
150
along a DNA strand + relates to moving _______ and - relates to moving ______
downstream; upstream
151
-10 sequence Pribnow box
TATAAT
152
in E. coli, 2 DNA sequences that act as a promoter are what 2 sequences
Pribnow box -10 and -35 sequence
153
how does the sigma subunit work
by decreasing the affinity of the polymerase for DNA, the sigma subunit allows the enzymes to rapidly scan the DNA for a promoter
- once the promoter is located, the subunit dissociates from the enzyme
154
Rho-independent (intrinsic termination)
RNA transcript forms a stable hairpin turn (stem-loop); RNA transcript contains a string of U's (stabilizer)
155
Rho-dependent (protein dependent)
rho factor binds to a C-rich region; contains helicase activity (energy from ATP hydrolysis) unwind the 3' end of the transcript from template; displace the DNA template strand
- break H-bond to separate RNA from DNA
156
transcription activators bind to the ___________
enhancers
157
RNA poly I
synthesis of the precursor of the large ribosomal RNAs
158
RNA poly II
synthesis of the precursors of message RNAs and snRNA inhibited by a-amanitin
159
RNA poly III
synthesis of small RNAs (tRNA)
160
eukaryotic gene promoter
RNA polymerase II
161
TATA, CAAT, and GC boxes and other cis-acting elements in eukaryotic promoters are recognized by
proteins other than RNA polymerase itself
162
TATA box
- centered at -25
- closely resembles the bacterial -10 sequence (TATAAT) but is further from the start site
163
start site
+1
164
CAAT box is located between
-40 and -150
165
what basal transcription factors help recruit and activate RNA polymerase II
CTF
SP1
TFIID
166
enhancers in eukaryotic gene tanscription
cis-acting elements that have no promoter activity of their own yet can exert their stimulatory actions over distances of several thousand base pairs
167
what are enhancers recognized by
transcription factors or tissue specific transcription factors
168
enhancer sequences are important for
establishing the tissue specificity of gene expression because a particular enhancer is effective only in certain cells
169
enhancer upstream vs downstream direction
upstream - left of gene
downstream - right of gene
170
what is considered the termination signal
Poly-A signal sequence
AAUAAA
171
posttranscriptional processing of rRNA
removal of external and internal transcribed spacers
172
what is the precursor in the nucleus that goes to 3 kinds of rRNAs
45S
173
posttranscriptional modification of tRNA three simple steps
a- cleavage (by RNase P)
b- additions (by tRNA nucleotidyl transferase)
c- modified nucleotides
174
mature form of tRNA
pseudouridine
175
reduction U->___
DHU
176
transversion U->___
psi
177
deamination A->____
I
178
how can RNA function as a catalyst
- RNase P cleaves the 5' end of pre-tRNAs
- composed of 12 kDa P protein and 400 nt long RNA
- catalytic activity lies within RNA part
179
ribozymes
- catalytic RNAs that function as enzymes and do NOT require proteins
- occur mostly within self-splicing introns and RNA encoded parasites
- limited to cleavage and ligation of RNA
180
angiozyme
ribozyme designated to inhibit vascular endothelial growth factor (VEGF)
181
heptazyme
ribozyme targets highly conserved sequences of the Hep C virus
182
first step of posttranscriptional modification of mRNA
5' capping: 7-methyl guanosine cap is added to the 5' end of the primary transcript by a 5'-5' phosphate linkage
- permit the initiation of translation and stabilize the mRNA
183
second step of posttranscriptional modification of mRNA
3' addition of a poly-A tail: (polyadenylation) is added to the 3' end of the transcript
- stabilize the mRNA and exit of the RNA from nucleus
184
third and final step of posttranscriptional modification of mRNA
removal of intron
185
how are introns and exons removed during modification of the mRNA
precise endonucleolytic cleavage and ligation reactions catalyzed by special splicing endonuclease and ligase (ribozyme)...then carried out by spliceosomes
186
spliceosomes
small nuclear ribonucleoprotein (snRNPs) recognize the splice sequence (intron begins with GU and ends with AG)
187
systemic lupus erythematosus
autoimmune disease, producing antibodies targeting snRNPs
188
B thalassemia
mutation at splicing sites (pGU---AGp) of the B globin resulting from a single nucleotide substitution in an acceptor splice site
189
RNA splicing in human diseases cause
reduced production of normal beta globin with poorly functioning abnormal beta globin
190
non-coding RNA
- molecule that functions without being translated into a protein; only 1.4% genome encoding proteins
- produce transcripts that function directly as structural, catalytic, or regulatory RNA
191
housekeeping ncRNAs
constitutively expressed and required for normal function and viability of cells
192
regulatory ncRNAs
expressed only in certain stages of organism development or as a response to external stimuli
- affect expression of other genes at transcription or translation
193
small interfering RNA (siRNA)
host immune response to restrict viral amplification
194
DCL/dicer
endoribonuclease, cleave double-stranded RNA and pre-miRNA into siRNA and miRNA
195
what is pri-miRNA transcribed by
RNA poly II
196
RNA induced silencing complex does what
has nuclease activity to degrade viral mRNA
197
siRNA-> RNA cleavage
double-strand RNA perfectly matched, only targets mRNA (coding region)
- natural antiviral defense in plants, fungi, and invertebrates
198
miRNA->translation inhibition
single-strand RNA some matched, more than one target non-coding region (3'-UTR)
- mechanism for regulation of gene expression
199
what mRNAs are subject to degradation
miRNAs that are not engaged in translation
- important in the regulation of gene expression (surveillance)
200
what are the steps to the degradation of mRNA
1- deadenylation by deadenylase
2- decapping by decapping enzymes
3- degradation by 3' to 5' exonuclease
201
three drugs that interfere RNA synthesis (intercalating agents)
Rifampin
Actinomycin D
a-amanitin
202
Rifampin
inhibits initiation and elongation
- blocks transcription by binding to the free polymerase
- inhibits elongation by binding to DNA bound polymerase
(TB)
203
actinomycin D
intercalates between the bases of DNA double helix, preventing the DNA from being used as a template
204
a-amanitin
synthesized by the poisonous mushroom amanita phalloides
- high specificity for eukaryotic RNA poly II
205
what intercalating agent is used for Hodgkins lymphoma
doxorubicin
206
specificity
particular codon always codes for the same amino acid
207
universality
the genetic code is virtually universal (exception in mitochondria)
208
redundancy/degeneracy
one amino acid might have more than one codon
209
nonoverlapping and commaless
starts with start codon (AUG)
210
sickle cell disease
missense mutation (codes for a different amino acid)
- glutamine turned into valine
211
Hemoglobin McKees Rocks
nonsense mutation (triggers early stop codon)
- tyrosine of the B chain is mutated to a stop codon (UAA)
- B hemoglobin's with shorter B chains have high oxygen affinity and decreased oxygen delivery
212
cystic fibrosis
phenylalanine is deleted in CFTR: ATP dependent transport protein, functions as cAMP regulated chloride channel
CFTR: cystic fibrosis transmembrane conductance regulator
213
components required for translation
- amino acids
- tRNA
- aminoacyl-tRNA synthetase
- mRNA
- small and large ribosomal subunits (factory)
- protein factors
- energy: 2 ATP equivalents and 2 GTP
214
in 2D all tRNAs appear as
cloverleaf pattern
215
amino acid accepting region on tRNA
acceptor stem - 3' CCA terminal region
- many of the nucleotides are involved in H bonds that form stems and loops
- amino acid is attached to a hydroxyl group of adenosine in the CCA region
- 5' end is phosphorylated and the 5' terminal residue is pG
216
anticodon of tRNA
in a loop near the center of the sequence
217
Wobble hypothesis
anticodon loop contains a sequence complimentary to the corresponding codon in a mRNA; some tRNAs can read more than one codon
- codons that differ in either the first two nucleotides must be recognized by DIFFERENT tRNA
- the first base of the anticodon determines the degree of the wobble...if the first base is inosine, the anticodon can recognize 3 codons
218
first base and last base of wobble hypothesis
C
A
U
G
I
G
U
A or G
U or C
U, C, A
219
aminoacyl-tRNA synthetase
attached AA to tRNA, has two activities
1- synthetase activity: the AA is first activated by reacting with ATP to form aminoacyl-AMP (adenylation) -- 2 high energy phosphate bonds
2- editing activity: the activated AA is transferred from aminoacyl-AMP to tRNA to form aminoacy-tRNA (charged tRNA) -- fidelity of translation is maintained by synthetase
220
what does initiation require the cooperation of
ribosomes
tRNA
mRNA
initiation factor proteins
221
what are the three binding sites of tRNA on the ribosome in protein synthesis
A (aminoacyl) site - binds incoming tRNA
P (peptidyl) site - binds tRNA with growing peptide chain
E (exit) site - binds uncharged tRNA before it leaves the ribosome
222
the _________ end of the tRNAs in the ___ site and ___ site are near one another at a site on the ____ subunit where the peptide bond is formed
acceptor
A site and P site
50S
223
initiation steps in protein synthesis (prokaryotic)
binding of mRNA to 3' end of 16S rRNA in 30S ribosomal subunit
- begins at least 25 nucleotides from the 5' end of the mRNA
- Shine-Delgarno sequence (in UTR) between the 5' end and the first codon translated
- directing the protein synthesis machinery to the start site and positioning the AUG codon in the P site
- 50S subunit joins the 30S subunit to form 7-S initiation complex
224
what form of methionine is present in bacteria
N-formyl-Met
225
elongation steps in protein synthesis (prokaryotic)
- elongation factors direct the binding of the appropriate tRNA to the codon in the empty A-site
- peptidyltransferase transfers the amino acid (peptide chain) from the P-site onto the amino acid at the A-site, and catalyzes peptide bond formation (ribozyme)
- ribosome moves 3 nucleotides along the mRNA in the 5' to 3' direction
- steps repeated until growing peptide complete
two high energy bonds
226
termination steps in protein synthesis (prokaryotic)
- termination codon is at the A-site
- release factors: RF-1: UAA, UAG, RF-2: UGA, UAA, RF-3: binds GTP; eRF
- recycle of the ribosomal subunit, mRNA, tRNA
one high energy bond
227
cleavage of how many high energy bonds are required per amino acid addition to the polypeptide
4
228
polyribosomes
several ribosomes can translate an mRNA molecule at the same time, forming polyribosomes or polysomes
229
how do eukaryotes differ in initiation of protein synthesis compared to prokaryotes
- formation of the preinitiation complex (PIC) with the 40S ribosome and met-tRNA with eIF-2
- eIF4E binds to the 5' cap of the mRNA and facilitates binding of PIC to mRNA
- PIC binds and searches for AUG
- 60S subunit then added to form 80S initiation complex
BINDS, THEN LOOKS FOR AUG
PROKARYOTES LOOK FOR AUG, THEN BIND
230
protein synthesis on the free ribsosome
- synthesized in cytoplasm
- completed protein delivered to intracellular location POST-translationally
- nucleus, chloroplast, mitochondria, peroxisomes
231
protein synthesis on the ER-bound ribosome
- directed through secretory pathway into the endoplasmic reticulum
- secreted proteins, Golgi, lysosomes, integral membrane proteins of organelles
232
protein synthesis steps on the RER (eukaroytic)
- translation of protein begins in cytosol
- as signal peptide emerges from ribosome, an SRP bind and inhibits further synthesis
- SRP is released and protein synthesis resumes
- signal peptide moves into the RER, signal peptidase removes
- synthesis of the nascent protein continues, completed protein is released into the lumen of the RER
233
SRP
signal-recognition particle
- inhibits translation
- docks the ribosome on the RER
234
what four targets could each protein that is synthesized on the RER go to
- RER resident
- Golgi resident
- plasma membrane resident
- endosomes/lysosomes
- extracellular space
235
what is the path that RER synthesized proteins take when targeting
1- travel in vesicles to the CIS face of the Golgi complex
2- membranes fuse, proteins in Golgi begin to bud and form the TRANS face of the Golgi complex in vesicles
3- vesicles may become lysosomes or secretory vesicles depending on their contents
4- secretory proteins are released from the cell when they fuse with the membrane
5- proteins with hydrophobic regions embedded in the membrane of the secretory vesicles may become cell membrane proteins
236
streptomycin
initiation stage
- binds to the 30s subunit of ribosome and interferes with the binding of fMet-tRNA
- thereby inhibits protein synthesis initiation in bacteria
237
tetracyclines
elongation stage
- interact with small ribosomal subunits, blocking access of the aminoacyl-tRNA to the mRNA- ribosome complex (prokaryotes)
238
puromycin
elongation stage
- resembles aminoacyl-tRNA becomes incorporated into the growing chain
- releases uncompleted polypeptide chains from the ribsosome
239
chloramphenicol
inhibits prokaryotic peptidyltransferas by binding to the 50S subunit
240
clindamycin and erthromycin
elongation stage
- binds to a site on the 50s subunit of the bacterial ribosome
- inhibiting translocation (prokaryotes)
241
Diphtheria toxin
elongation stage
- inactivate the eukaryotic elongation factor (eEF-2)
- preventing translocation (eukaryotes)
242
What does Risin do to the 28S ribosomal RNA
- in cytosol, ricin A-chain inactivates ribosomes by depurination pf 28S rRNA in 60S ribosomal subunit
- a Ricin removes adenine from position 4324 by its N-glycosidase activity
- adenine A4324 site ricin A-chain rich
castor beans
removes the purine 'A' in the loop
243
Ricin
inactivates ribosomes by depurination of 28S rRNA (peptidyltransferase) in 60S ribosomal subunit
244
types of posttranslational modifications of proteins
trimming:
1- inactivate precursors
2 - zymogens
covalent modifications
245
what type of posttranslational modification of proteins is the formation of human insulin from preproinsulin
trimming
- 3 disulfide bonds synthesized in the ER
246
what type of posttranslational modification of proteins is the zymogen activation by proteolytic cleavage
trimming
activation cascade
247
types of covalent modifications in posttranscriptional modification of proteins and the AA they combat
phosphorylation (Tyr, Ser, Thr) - metabolic
regulation, signal transduction
hydroxylation (proline) - in collagen, ER
glycosylation (O linked)
other - biotinylation, farnesylation, prenylation, ubiquination
248
how are histone tails subject to a variety of modifications
- the histone code hypothesis proposes that specific combinations of modifications, as well as the order in which they occur, help determine chromatin configuration and influence transcription
249
ubiquitin proteasome pathway
unneeded proteins can be tagged with the protein ubiquitin (76 amino acids) at the lysine residues
- protein is chopped up by proteosomes
250
what forms the 26S proteasome
19S regulatory particle
20S proteasome (proteolytic)
19S regulatory particle (substrate recognition)
- large ATP-dependent protease and degrades polyubiquitinated proteins
251
what gene is linked for ubiquitination degradation
K48
252
why do prokaryotes and eukaryotes alter gene expression
in response to their changing environment
253
what is the main site of control for genes
transcription
254
in multicellular eukaryotes, what does gene expression do
regulate development and is responsible for differences in cell types
255
what is the Lactose operon of E. coli
bacteria rely on glucose as their source of carbon and energy
- when glucose is scarce, E. coli can use lactose as its carbon source
256
what genes in the lactose operon are used with what enzymes
lacZ - B-galactosidase
lacY - galactoside permease
lacA - thiogalactoside transacetylase
257
the genes lacA, lacY, and lacZ are transcribed from a _______ _________ that produces a single mRNA from which the 3 proteins are translated _________
single promoter; polycistronic mRNA (monocistronic in prokaryotes)
258
the lactose operon is regulated by what two elements
operator - suppressed by Lac repressor - repressor inhibits transcription by binding to the lac operator (prevented by the inducer/lactose sensors)
CAP binding site - activated by catabolite activator protein (CAP), stimulates transcription of lactose gene when bound to cAMP/glucose sensor
259
in order to have transcription in the lactose operon what two things must happen
- remove lactose repressor
- bind CAP
260
when there is + glucose and - lactose in the lactose operon what is happening and what is inhibited by glucose not allowing cAMP to be made
operon off
adenylyl cyclase inhibited by glucose
261
when there is - glucose and + lactose in the lactose operon, what is happening and what binds to the repressor protein
operon on
- allolactose binds to repressor protein, causing a conformation change that prevents it binding to the operator
- operator is not blocked and RNA poly can initiate transcription
262
when there is + glucose and + lactose in the lactose operon, what is happening
operon off
- if there is glucose, CAP will not bind to cAMP so RNA polymerase cannot initiate transcription
263
when there is - glucose and - lactose in the lactose operon, what is happening
operon off
- even though CAP can bind because of glucose absence, there is no lactose, so the repressor binds to the operator (cannot be released)
264
transcription factors
- proteins that bind to specific DNA sequences
- control the genetic info from DNA to mRNA
- perform alone or with proteins in a complex (promoting - activator) or (blocking - repressor) the recruitment of RNA polymerase to specific genes
- contain at least one DNA-binding domain, and a domain to activate or inhibit transcription
265
cis-acting elements in transcription
enhancer (upstream/downstream)
promoter proximal elements
DNA sequences that regulate the expression of a gene located on the same molecule of DNA
266
transcription (trans-acting) factors
basal transcription complex
regulatory DNA binding proteins (specific)
proteins that recognize cis-acting elements and regulate RNA synthesis
267
oncogene transcription factors
RARa
c-myc
Fos, jun
268
tumor suppressor transcription factors
p53
RUNX3
269
gene expression regulation in lipid soluble hormones
- hydrophobic signal molecules
- go through plasma membrane and go into cells and bind to corresponding receptors
270
gene expression regulation in water soluble hormones
(insulin, glucagon)
- deliver signals outside the cells by binding to receptors on cell membrane
271
transcription by lipid soluble hormones
- go through plasma membrane and bind to hormone receptors; go directly into nucleus
- hormone receptors bind to specific regions of DNA called hormone response elements (HRE) - enhancer - and activate gene expression
- nuclear hormone receptors (TF) have two highly conserved domains
272
what are the two highly conserved domains of nuclear hormone receptors in lipid soluble
- DNA binding domain
- ligand binding domain
273
CREB
cAMP response element binding protein
274
transcriptional regulation by cell surface receptors (water soluble hormones) first and second messengers
first: insulin, epinephrine, glucagon
second: cAMP, cGMP, calcium
- CREB phosphorylates the transcription factors and either inhibits or promotes glycogen synthesis
275
gene regulation at post-transcription methods
alternative splicing
mRNA editing
mRNA stability
276
alternative splicing
- recognizes different splice sites in different tissues (introns spliced out of pre-mRNAs)
- results in different polypeptide products from same gene
- increases functional diversity and complexity of an organism, encodes more than 100,000 proteins
- factor in genetic diseases and strongly associated with protein disorder
277
assembly and secretion of chylomicrons by intestinal mucosal epithelial cells
- newly synthesized TG and cholesterol ester are packed as lipid droplets surround by thing layer of:
apolipoprotein B48 (apo B-48)
phospholipids
free cholesterol
278
RNA editing of apo-B pre-mRNA
1- deamination of cytidine to uridine by cytidine deaminase (C to U) by oxidative deamination apoB-48 --> non-sense mutation
2- adenosine to inosine (A to I) adenosine deaminases acting on dsRNA
279
two methods of mRNA stability
iron metabolism
RNA interference (siRNA and miRNA pathways)
280
microRNA (miRNA)
small pieces of genetic information that play a crucial role in switching genes on and off
281
Transferrin receptor (TfR)
absorption
- carrier protein
- needed for the import of iron into the cell and is regulated in response to intracellular iron concentration
- imports iron by internalizing the transferrin-iron complex
- receptor-mediated endocytosis
3' UTR end - stabilizes mRNA when added only increasing translation
282
Ferritin
storage
- protein that stores iron, releasing it when the body needs it
5' end prevent translation initiation when binded allowing only for storage
283
the production of transferrin and ferritin is regulated where
the level of mRNA by iron regulatory proteins (IRPs), which bind to iron response elements (IREs)
284
transferrin receptor decreases
mRNA stability
285
ferritin increases
protein translation
286
epigenetics
study of heritable changes in gene expression that does not involve changes to the underlying DNA sequence - affects how the cells read the genes
287
chromatin
eukaryotic DNA packaged with proteins
288
epigenetic regulation at expression pathways
- posttranslational modifications of histone proteins
- DNA methylation
289
heterochromatin vs chromatin
heterochromatin (closed)
- inactive
- not usually expressed
- modification of the N-terminal tails of histones alters chromatin accessibility of gene expression
euchromatin (open)
- active
- chemical modifications to DNA of chromatin influences both structure and expression
290
HAT and HDAC
HAT: histone acetyltransferase
HDAC: deacetylase
291
reversible acetylation and gene expression
- lysine bears a positively charged ammonium group at neutral pH
- addition of acetyl group neutralizes ammonium group to an amide group while adding a negative charge
acetylation by HAT (transcription activation)
deacetylation by HDAC (gene silencing)
charge dramatically reduces the affinity of the tail for DNA and entire histone complex, loosening from the DNA
292
DNA methylation
DNMT: DNA methyltransferase
- occurs on cytosine 5 position of CpG nucleotides
- DNA methylation is associated with reduced transcription
- cause long-term inactivation of genes
293
DNA methylation and cancer
hyper: promoter silencing
deamination: meCpG -> meTpG mutation
both block tumor suppressors
294
fragile X syndrome
single most common form of inherited mental retardation
- hypermethylation on C followed by G in promoter region of FMR1 gene
- no mRNA, no FMRP protein
295
5-Azacytidine
DNA methyltransferase inhibitor
- converted to the deoxynucleotide triphosphates, incorporated in place of cytosine in replicating DNA
- DNMTs get trapped
- resulting in formation of heritably demethylated DNA
(myelodysplastic syndrome)
5' C becomes taken by N and DNMT has no where to synthesize leaving it trapped
296
three techniques that facilitate analysis of human DNA
- obtain fragment of DNA and copies of genes
- amplify DNA sequences or genes
- Identify DNA sequences or genes
297
Palindrome
when read in the 5' to 3' direction, the sequence on the top strand is identical to that of the bottom strand
298
EcoRI
palindrome genus of the bacterium, type/strain of the bacterium, or order of the discovery
299
restriction endonucleases
TaqI - four base, cohesive ends
Hae III - four base, blunt ends
300
what are key tools for forming recombinant DNA molecules
restriction enzymes and DNA ligase
301
how is a complementary DNA duplex created from mRNA
using reverse transcriptase
- cDNA does not contain introns because mRNA (no introns) is used as a template
- cDNA lacks the regulatory regions of a gene
302
DNA cloning
cloning vectors: used to accommodate the DNA of interest
- put DAN into vector and get amplified response
- relies on restriction enzymes
303
T/F during cloning, bacteria are grown in the presence of antibiotics
true
304
polymerase chain reaction - PCR
1- template
2- primers
3- dNTP
4- thermostable DNA polymerase
- denature DNA into separate strand by heat
- anneal primers
- extend primers
305
when is Taq DNA polymerase used in PCR
after the first cycle on the target sequence
(Thermus acquitis)
306
gel electrophoresis
fragment separation
- phosphodiester backbone negatively charged
- shorter DNA travels farther
- stain with ethidium bromide
307
southern blotting
detection of a specific DNA sequence
- combines electrophoresis to a filter membrane and subsequent fragment detection by probe hybridization
308
southern blotting steps
- separation of DNA fragments
- denaturing DNA (alkali solution)
- ssDNA probe hybridizes only with complementary sequence on DNA
309
DNA sequencing
nucleotide by nucleotide identification
- dideoxyribonucleoside triphosphate prevents strand extension at 3' end
310
difference between southern, northern, and western blot
southern - detect DNA
northern - detect RNA
western - detect protein (antibody probe)
311
real-time PCR (qRT-PCR)
monitor the progress of a PCR reaction in real time
- detection of the fluorescence produced by a reporter molecule that bind to a dsDNA (SYBR green intercalating)
- higher intensity of dye is higher numbers of DNA
312
T/F before RNA sequencing, you must convert RNA fragments into cDNA
true
312
microarray and spot colors
contain thousands of immobilized DNA sequences (simultaneously analyzed)
dark - neither cell produces
red - cancer produces
green - normal produces
yellow - both cells produce
313
denaturing proteins by SDS
sodium dodecyl sulfate
- disulfide bond reducer is 2-mercaptoethanol
- proteins separated by mass
314
how does a western blot work
- stained with fluorescent labeled antibody that gets excited by UV light and the corresponding band to the protein binds which antibody
315
Horseradish peroxidase (HRP)
stain in western blotting that reacts with a chemiluminescent HRP substrate and light is produced
316
Enzyme-linked immunosorbent assay (ELISA)
- blood samples containing Covid-19 viral proteins (spike) binds to the antibody and converts to colored product (chemiluminescent)
317
monoclonal antibody drugs
anti-spike Ab
- stops binding and infection
- blockade of entry
318
remdesivir
nucleoside analog targets RNA polymerase
- activity inhibited
- blockade of replication
319
corticosteroid
dexamethasome anti-inflammatory
- overproduction of cytokine storm in inhibited and reduce the number
- innate immune response/enhancers of antiviral innate immunity
320
paxlovid
combo therapy drug of nirmatrelvir and ritonavir
- protease inhibitor
321
protein subunit vaccine
spike protein is combined with substances to boost immune response
322
viral vector vaccine
genetic material inserted into inactive virus and make own spike proteins and initiate
323
mRNA vaccine
gives cells instructions on how to make spike proteins and begin displaying them for antibody response
324
genome editing
ability to change an organisms DNA
- added, removed, altered at particular locations
325
CRISPR - clustered regularly interspaced short palindromic repeats
adapted in editing system in bacteria to fight against viruses
- Cas9 an RNA-guided DNA endonuclease enzyme
- PAM 2-6 base pair DNA sequence
MCB 450
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