Midterm 1 Flashcards
1
Q
Name of bond that links Sugar to Base
A
B-N-Glycosidic Bond
2
Q
Primary Structure of Nucleic Acids
A
Linear chains of interconnected Nucleotides forming a backbone
(Phosphodiester bond)
3
Q
Secondary Structure of Nucleic Acids
A
Double helix where base pairing occurs between 2 strands and H-Bond linkage
4
Q
A-DNA
A
- Right Handed
- 2.6nm diameter
- 11 Bp/turn
- Anti conf.
5
Q
B-DNA
A
MOST COMMON
- Right handed
- 2.0nm diameter
- 10 Bp/turn
- Anti conf.
6
Q
Z-DNA
A
- Left handed
- 1.8nm diameter
- 12 Bp/turn
- Pyr: Anti , Pur: Syn
7
Q
Tautomers
A
Isomers differing in position of Proton or Electron
- Oxo-Enol
- Amino-Imino
8
Q
What are the physiologically preferred Tautomer forms of Bases
A
Oxo (=o) & Amino (NH2)
9
Q
2 Types of Heterochromatin
A
- Facultative: Can become Euchromatin
- Constitutive: Perm. condensed
10
Q
DNA Condensation forms
A
1) DNA Double Helix (2nm)
2) Beads on a String (11nm)
3) Solenoid Coil (30nm)
4) Radial Loop (300nm)
5) Chromatin (700nm)
6) Metaphase Chromosome (1400nm)
11
Q
Histones that form Histone protein core
A
- H2A
- H2B
- H3
- H4
12
Q
Chromatosome
A
Nucleosome bound to a H1 histone protein and adjacent Linker DNA
13
Q
Non-Histone Proteins
A
- HMG (high mobility group)
- SMC (struct. maintenance of chromosome)
14
Q
Condensin
A
Type of Structural maintenance of Chromosome (SMC) that extrudes and stabilizes DNA
(Activated by CDK)
15
Q
What phase do we usually find chromatin in?
A
Interphase
16
Q
Superspirilization in Prokaryotes
A
- Positive Superspiralization (Overwound DNA)
- Negative Superspiralization (Underwound DNA)
17
Q
Linking Number
A
Number that describes linking of 2 closed circular DNA
Twists + Writhes
18
Q
Twists
A
Right-handed helical turn
19
Q
Writhes
A
Suprahelical turn in Negative Left-handed orientation
(unwinding a twist leads to writhes)
20
Q
Topoisomerases
A
Enzymes that relieve Torsional stress during DNA replication
Tyrosine active site
21
Q
Type I Topoisomerase
A
- Acts on 1 strand only
- No energy
- Changes linking n. by 1
- Creates nick
(only for negative supercoiling in Prokaryotes)
22
Q
Type II Topoisomerase
A
- Acts on both strands
- Requires ATP
- Changes linking n. by 2
- e.g DNA Gyrase
23
Q
Human Genome
A
All genetic information in a cell which includes Nuclear and Mitochondrial DNA
(apx. 3 Billion BP)
24
Q
Length of the Human Genome
A
2m
25
Mitochondrial Genome
Circular DNA with No Histones, No Introns!
37 total genes, 13 protein coding.
22 tRNA
2 rRNA
(mRNA is Polycistronic)
26
Polycistronic
One coding region can code for more than one peptide
(only in prokaryotes/mitochondria)
27
What does Mitochondrial DNA lack?
5' Cap
28
5' Cap
Modified Guanosine neuc. added to 5' end of mRNA in Eukaryotes
29
What determines the complexity of the Human Genome?
The number of non-coding regions (introns)
30
Gene
Entire nucleic acid sequence that is necessary for the synth. of a functional gene product
31
What are Eukaryotic genes made up of?
- Promoter region
- Open Reading Frame (ORF)
32
Promoter region function
- Initiates Transcription
- Binds RNA Polymerase
33
2 Parts of the Promoter region
1) Core promoter
3) Proximal promoter
34
Core promoter
- Region initiating transcription
- Initiator element (Inr) with Start codon
- DPE & MTE for preinitiation complex
- TATA Box
- B-recognition element (BRE)
35
Downstream Promoter Element (DPE) function
Binds Factor II D which helps recruit RNA Polymerase
36
TATA Box
- Rich in Thymine and Adenine
- Defines direction of Transcription
- Recognized by TBP, TFIID
- (-25)
37
B-recognition element (BRE)
- Upstream of TATA box
- Binds Factor II B, to start transcription (starting point)
38
Initiator element (Inr)
- Recognized by TFIID
- Near transcription site
- Can sub. TATA
39
Proximal Promoter
- Upstream of Core Promoter
- CAAT Box
- GC Box
40
CAAT Box & GC Box
- Binds regulatory proteins or transcription factors (enhancers)
- (-80) CAAT
- (-100) GC
41
Open Reading Frame (ORF)
Region of the gene that is transcribed to an RNA molecule from START to STOP codons
42
How do Enhancers/Silencers interact with Promoter region?
Formation of a DNA loop by folding so that e/s is in close prox. to the promoter region
43
Mechanisms of Enhancer Regulation
1) Rigid Model (Enhanceosome)
2) Flexible Model (Billboard)
3) Collective Model
44
3 Types of Exons
1) Initiator Exon (splice site + start)
2) Internal Exon (2x splice site)
3) Terminal Exon (splice site + stop)
45
Alternative Splicing
Process that allows single gene to code for multiple proteins
So exon can be intron of other protein (vice versa)
46
Insulators
Regulatory gene sequences binding insulator proteins
47
Types of Insulators
- Barrier Insulators
- Enhancer Insulators
48
Barrier Insulators
Sequence before a gene preventing heterochromatization, allowing transcription
49
Enhancer Insulators
Sequences between enhancers and promoters to stop DNA loop formation
(No interaction of promoter & e/s)
50
Psudogenes
DNA sequences resembling a functional gene but have MUTATIONS that prevent proper expression
(Gene-Like structures)
51
Pseudogene functions
- Can code for Pgk2
- Used as template for transcription (non-coding)
- 3D chromatin interaction (stabilize DNA)
- Gene conversion (rare)
52
Direct Pseudogene formation
- Gene mutation (Point)
- Non-processed pseudogene
- e.g L-gulonolactone oxidase showing humans were once able to produce VitC (gene fossil)
53
Indirect Pseudogene formation
Reverse transcription of mRNA/RNA back to DNA inserting into chromosomal DNA (processed pseudogene)
54
Transposons
Segments of DNA that can move around to different positions in the genome of a single cell
(jumping genes)
55
Class II Transposon
- DNA Transposon
- Sequence cut and pasted to new place
- Transposase enzyme used
56
Class I Transposon
- Retrotransposon
- Copy and paste but RNA (RNA poly)
- RNA then back to DNA (reverse transcriptase) then integrated (Integrase)
57
Types of Interspersed Repeats
- LINEs
- SINEs
- LTR Elements
58
LINEs
Long Interspersed Nuclear Element
- Retrotransposon
- Codes for ORF1 (p40 mRNA) & ORF2 (endonucleases & rev. transc.)
59
SINEs
Short Interspersed Nuclear Element
- Parasite transposed element using LINE elements to multiply (ORF1/2 & reverse transcriptase and endonuclease)
60
LTR Elements
Long Terminal Repeat Elements
- Retrotransposons
- Found at end of retrovirus genome
- For integration of Virus into host DNA
61
What virus is integrated in Human genome
HERV
Human Endogen Retrovirus
8% integration
62
Tandem Repeats
Repeat sequences one after the other with no breaks or bases in bw
Formed by DNA slippage
63
Types of Tandem Repeats
- Microsatellite (1-9 bp)
- Minisatellite (10-100 bp)
- Macrosatellite (100+ bp)
64
What causes Fragile X Syndrome
Mutations in FMR1 Gene
(CGG repeat)
65
Type I DNA Dependent DNA Polymerase
- Synthesis, 3' & 5' Exonuclease activity
- Particularly works on Lagging strand
- Role in repair
66
Type II DNA Dependent DNA Polymerase
- DNA Repair
- Synth & 3' Exonuclease
- Not required for DNA replication
67
Type III DNA Dependent DNA Polymerase
- Replication
- α, ε, and θ Subunits for synth.
- 2 B subunits for attachment
- Synth & 3' Exonuclease
68
OriC
Single defined replication origin in Prokaryotes
69
DNA separation in Prokaryotes
- dnaA recognize OriC and bind dnaA box
- dnaB & dnaC form Helicase-like complex
70
Primosome
Group of proteins that help initiate DNA replication by setting up proper conditions needed for synthesis
71
Parts of the Primosome
- HD-proteins: Keep DNA single stranded
- N-proteins: attached to dnaB & dnaC
- Primase / dnaG: Synth. RNA primer
72
Replisome
Combination of Primosome and DNA polymerase III
73
What does DNA ligase use for energy in Prokaryotes?
NAD +
74
What happens in G1?
1) ORC to starting point on DNA
2) CdC6 & Cdtl proteins form complex similar to dnaB/C (helicase)
3) Mcm form pre-initiation complex, active helicase
75
What inhibits mcm proteins?
CdC6 and Cdt1 because they are the ones who recruit it
76
Cyclins
Family of regulatory proteins
- Can switch on specific CDKs
- CDKs phosphorylate proteins
77
What happens in S phase?
1) Cdc6 phosphorylated by CDKs
2) Mcm not inhibited anymore by cdc6
3) Helicase activity
4) ORC phosphorylated and inhibited to prevent re-initiation
78
α - DNA Polymerase
Only synthesizes initial DNA segment
- Extends RNA primer by 25-ish nucleotides (mixed RNA-DNA primer)
- Forms a tetramer complex with Primase
79
β - DNA Polymerase
DNA Repair
(similar to DNA polymerase I in prokaryotes)
80
γ - DNA Polymerase
DNA synthesis in the Mitochondria
81
δ - DNA Polymerase
DNA synthesis on Lagging strand
82
ε - DNA Polymerase
DNA synthesis on Leading strand
83
PCNA
- Sliding clamp
- Ensures that δ and ε DNA polymerases dont dissociate from template
- Increases processivity of the polymerase
84
RFC
Clamp Loader
Inserts and opens PCNA ring to encircle region of DNA synthesized by polymerase alpha
85
What removes Ribonucleotides at 5' end of Okazaki fragments
- Ribonuclease H
- FEN I
(5' exonuclease activity)
86
Telomerase
Enzyme that adds telomeric repeat sequences to 3' ends of each chromosome to compensate for shortening due to RNA primer removal on lagging strand
87
Hayflick limit
Limit of cell division before the telomere is too short
(Ageing)
88
What stabilizes the T-loop
Shelterin complexes
89
Shelterin complexes
- TRF 1 / 2 (inh. telomerase)
- TIN 2
- TIPP 1 (activates telomerase)
- POT 1
90
POT 1 and TRF 2 significance
Sheltrin proteins that inhibit ATM and ATR which are kinases that recognize telomere as damage
91
p53
Tumor supressor protein
- Checks DNA during G1 before replication
- All corrected before S phase
92
Common types of DNA damage
- Thymine dimers
- Modification of bases by exo and endo agents
- Loss of Purine base
- Deamination
93
DNA intercalators
Modify structure of the double helix causing breakage and stoppage of replication
94
What types of DNA damage happen spontaneously?
- Loss of a Purine base
- Deamination
95
Direct Repair
Damage is identified and corrected
(present in prokaryotes to repair Thymine dimers using photolyase)
96
Base Excision repair
Damaged base is removed and missing part of the chain is resythesized
(Deamination and loss of purine base repaired)
97
Nucleotide Excision repair
Entire DNA segment with the false conformation is removed , then resynthesized
(Repair of thymine dimers)
98
Mutation
Permanent alteration in DNA (<1%)
99
Types of chromosome mutations
- Deletion, Insertion, Repeats
- Inversion
- Translocation
100
Polymorphism
Genetic variation that is present in opulation with high allele freq. (>1%)
101
DNA Deamination
Loss of an amino group from Bases of DNA, generating a foreign base.
- Oxidative deamination
- Keto group replaces amino
- Cytosine most common
102
Deamination of Adenine
Hypoxanthine
(pairs to C)
103
Deamination of Guanine
Xanthine
(pairs to C)
104
Deamination of Cytosine
Uracil
(pairs to A)
105
Repair of DNA Deamination
Base Excision Repair
1) Glycosidase enzyme removes deaminated base (AP site)
2) AP endonuclease removes sugar
3) DNA polymerase I/B resynth
4) DNA ligase adds ester bond
106
Thymine dimers
2 Thymines break their double bonds with Adenine and form 2 single bonds with each other
(also C-C or C-T)
107
What causes Thymine dimers
UV light
by electron excitation
108
Why is Thymine dimer bad
2 Thymines will be read as 1, so only 1 adenine is added to the strand
(replication and transcription inhibited)
109
Repair of Thymine Dimers
Nucleotide Excision Pair
1) Specific endonuclease used to remove whole segment
2) DNA polymerase I/B resynth
3) DNA ligase adds ester bond
110
Difference between base and nucleotide excision pair
Glycosidase is used first in a base excision
in Nucleotide excision, we use a special endonuclease only
111
Repair of Thymine Dimers in Prokaryotes
Direct repair
Photolyase enzyme which can reverse dimerization
Using UV light
112
DNA mismatch
DNA damage caused by incorrect pairing of 2 bases on double helix
113
Repair of DNA mismatch, how do we know which base is incorrect?
During synthesis, the template strand is methylated and the synthesized isnt methylated for a while
114
Repair of DNA mismatch
1) MutS2 recognizes the mismatch
2) MutH endonuclease attaches to wrong base (inactive)
3) MutL2 activates MutH to cut region of unmethylated DNA, & UvrD helicase
4) Exonuclease breaks strand from hemi-methylated region to mismatch
5) DNA polymerase III and Ligase
115
Repair of DNA mismatch, why dont we use DNA polymerase I?
Because its too slow
DNA Polymerase III is needed to synthesize a long segment
116
Point Mutation
Base change with a low allele frequency (rare)
117
Single Nucleotide Polymorphism (SNP)
Base change with High allele frequency (common)
118
Same sense, Synonymous, Silent mutation
Nothing happens to the amino acid sequence
119
Missense Mutation
Change of one of amino acids
120
False splicing
- If one Intron remains after splicing, mRNA is longer than it should be.
- If one Exon is missing then part of the protein is missing
121
Nonsense mutation
Amino acid codon is replaced by a stop codon
Truncated protein (shorter than it should be)
122
Frameshift
When a base is inserted or deleted from the coding sequence causing shift.
(can also cause a nonsense mutation)
123
Diseases caused by Trinucleotide repeats
- Fragile X Syndrome
- Myotonic Dystrophy
- Huntington disease
124
Diseases caused by Point Mutations
- Sickle Cell Anemia
- PKU
125
Diseases caused by SNP
- Cystic Fibrosis
126
Huntington Disease
CAG repeat (poly-glutamine in protein)
Proteases cant degrade proteases
(neurodegenerative)
Autosomal Dominant
127
Sickle Cell Anaemia
Point Mutation of Hb B-gene
Glu to Val change
Altered surface molecules
Recessive allele
128
Phenylketonuria
Mutation of Phenylalanine Hydroxylase
Cofactor Deficiency (BH4)
129
Cystic Fibrosis
CFTR protein mutation
Nonsense Mutation
Autosomal Recessive
(Genetherapy, adenovirus containing CFTR gene)
130
Methods of Identifying Genetic Factors
- Genome wide association study (GWAS)
- Candidate Gene Analysis
- Case-control Study
- Transmission disequilibrium test
131
Genome Wide association study (GWAS)
- Polymorphisms are in the whole genome
- No hypothesis needed
- Stats. analysis for correction of multiple tests
132
Candidate Gene Analysis
- Hypothesis set before analysis
- Selected genes analyzed
- Sometimes important targets can be left out
133
Case Control Study
Allele or Genotype frequencies of Case and Control groups compared to check for difference.
(GWAS & Candidate used)
134
Transmission disequilibrium test
- Affected child is studied to see what allele came from what parent
- Only heterozygote parents included in the study
- If there is a monogenic disorder they will be effected
- Compares rates of alleles transmitted and untransmitted to the affected offspring from the parents
135
What does PCR stand for
Polymerase Chain Reaction
136
PCR is...
Artificial method of replicating DNA in lab
It is a form of in vitro DNA rep.
137
PCR vs Normal replication
PCR only a small part of the genome is replicated, while in normal the whole genome is replicated
138
How do we separate strands in PCR?
High temperature only, no proteins needed
(denaturation step)
139
How many cycles of replication in PCR?
35 - 40 semi-conservative replication cycles in a Thermocycler
140
3 steps of PCR
1) Denaturation
2) Annealing
3) Elongation
141
PCR Denaturation
90 - 95 degrees
Separation of Strands
Only hydrogen bonds break
142
PCR Annealing
Cooled to 50 - 72 degrees
25 - 30 seconds
Allows Primers to Anneal/Bind
143
Primers in PCR
Are DNA!!!
ssDNA (16-30) synth. in test tubes
144
How do we determine the Annealing Temp?
Based on the melting temperature of the ssDNA primer
Too High: no H-bond formation
Too Low: non-spec. binding of primer
145
PCR Elongation
Heated to 68 - 72 degrees
Optimal temperature for DNA polymerase
Taq DNA polymerase used as it is thermostable
Keeps elongating till Denaturation temp is reached
146
When do we get the correct sized product in PCR?
When the synthesized strand from the 1st cycle is used as a template.
Using original DNA causes overhanging of strand.
147
Components of PCR reaction mixture
- DNA template
- ssDNA oligonucleotide primer
- dNTPs
- Taq DNA polymerase (therm.stab)
- Buffer (for optimal env.)
148
What do we use to see PCR results?
Agarose Gel Electrophoresis and DNA ladder to compare.
+ Intercalator dye (Ethidium Bromide)
149
Novel Polymorphism
Genetic variation in DNA that has not been previously characterized or documented
150
Sanger Sequencing
Direct detection of nucleotide sequence
1) Primer added to DNA strand of interest
2) DNA poly. extends the primer
3) Both dNTP and specific ddNTP incorporated to see where it binds
4) When ddNTP, no 3' OH group so no ester bonds formed so it stops
5) Different length DNA fragments to be separated by size
151
Next Generation Sequencing (NGS)
One DNA sample is sequenced several times.
We can measure qualitatively by checking pH or PPi after each nucleotide added
PCR based method
152
Primer Extension Method - Mini Sequencing
'Mini' because we need 1 single primer
Only elongate primer by 1 nucleotide.
Color labelled ddNTPs, added complementary next to primer
153
Restriction Fragment Length Polymorphism (RFLP)
Changes a sequence difference to a length difference
C/T Polymorphism
Type II restriction endonuclease needed (highly specific)
T - perfecT recognition
Cut, length analyzed
154
Allele Specific PCR
Special DNA polymerase needed with no 3' exonuclease activity
- Primer ends ON the SNP not before
- if complementary it continues
- if not it stops
- 3' exo. would just cut the wrong base and keep going which defeats the whole purpose
155
Prokaryotic Transcription Strands
- Template strand
- Coding strand
156
Can transcription start without a primer in Prokaryotes?
Yes, because RNA polymerase is used
157
Proofreading in RNA polymerase?
No, no 3' exonuclease activity as RNA copy leaves the nucleus so no need, wont be genetically inherited
158
What is prokaryotic transcription unit split into?
- Promoter region (-10, -35)
- Transcribed region
159
2 forms of RNA polymerase
- Holoenzyme
- Apoenzyme
160
Holoenzyme RNA Polymerase
Core + σ subunit (5)
(2a, B, B', σ)
Can initiate transcription
Low affinity (1s) searching
161
Apoenzyme RNA polymerase
4 subunits (2a, B, B')
Can only elongate the RNA chain
High affinity (60min) holding
162
Role of σ Subunit in RNA polymerase
Initiates transcription by interaction with promoter region
163
Is helicase used in Prokaryotic transcription
No
RNA Polymerase can unwind helix
164
Inhibitor of RNA polymerase in Prokaryotes
Natural Rifamycin
Binds to B subunit and prevents initiation
165
Termination of Transcription types in Prokaryotes (2)
- Rho independent termination
- Rho dependent termination
166
Rho independent Termination
1) RNA pol. reaches termination sequence
2) Termination sequence forms hairpin loop (G-C), poly. slows
3) Loop followed by U rich sequence
4) RNA strand pulled out of pol. due to weak U H-bonds.
167
Rho dependent Termination
1) Rho (ρ) factor Helicase follows RNA polymerase
2) Uses ATP
3) RNA poly. slows down, Rho (ρ) factor catches up
4) Rho (ρ) factor pulls RNA strand out, because no U rich sequence after
168
Post-transcriptional RNA modifications in prokaryotic cells
RNA transcript has tRNA and rRNA coding segments which can be modified after being cut out by endonuclease (RNase)
e.g addition of CCA sequence to tRNA on 3' end
169
Operon
One transcription unit that can code for more than one protein
Only in Prokaryotes
170
Ribosome binding sites (RBSs)
Located upstream of Start codon
1st cistron RBS in 5'UTR, none in 3'
171
Activator / Repressor Regulatory Proteins
Bind to regions in the Promoter called Operator regions
172
Name of immature mRNA
premRNA or hnmRNA
173
Type II RNA Polymerase
premRNA to mRNAs
Can not initiate transcription
174
a-amanitin
Death cap mushroom poison that inhibits transcription by inhibiting RNA polymerase
175
Initiation of Transcription in Eukaryotes
1) TFIID binds TATA
2) TFIIA/B --> core prom. --> TFIIF brings RNA Polymerase II
3) TFIIH unwinds double helix at INR using ATP
176
Termiantion of Transcription in Eukaryotes
1) CPSF and CstF follow RNA polymerase II
2) CPSF detects stop codon (Poly-adenylation)
3) CPSF binds with high affinity to sequence and detaches from polymerase with help of CstF.
177
Exon def
Part of the gene that remains in the mRNA after maturation
178
5' cap formation
1) 1st nucleotide is usually ATP, RNA terminal phosphatase removes 1 phosphate
2) GTP added to ATP by Guanylyl transferase enzyme, PPi released
3) Methylated by methyltransferase enzyme (SAM donor)
179
5' cap function
- Maturation signal
- Protective measure (shields from 5' exonuc.)
- Initiation of Translation (elF4E)
180
Splicing
Splice sites bw introns and exons.
Inside Splicosome:
1) branching adenine attacks 5' splice site forming loop (lariat)
2) Then attacks 3' splice site linking exons together
181
snRNPs in Splicing
- U1: 5' splice site
- U2: Branching site (adenine)
- U5: 3' splice site
ALL FORM SPLICOSOME
182
Poly-A Tail formation
During termination of transcription
1) CSPF inactive following RNA polymerase II
2) Stop codon reached (TTATTT), AAUAAA transcribed on mRNA
3) CSPF recognizes and recruits CstF to cleave transcript
4) Polyadenylate polymerase enzyme add 150-200 adenine nucleotides
(Polyadenylate BP cover tail)
183
Transport of the ready mRNA outside of the nucleus
1) mRNA covered by proteins recognized by Nuclear export receptor that its mature
2) Works with Ran GTP BP to direct mRNA to nuclear pore
3) 5' Cap binding complex switched to Cytosolic cap binding proteins after exiting
4) Loop forms, Poly-A tail touches 5' Cap
