1-DNA & RNA Flashcards
(232 cards)
1
Q
Bond that links Sugar to Base
A
B-N-Glycosidic Bond
(1’ of sugar)
2
Q
A.A necessary for Purine synthesis
A
- Glycine
- Aspartate
- Glutamine
3
Q
Primary Structure of Nucleic Acids
A
Linear chains of interconnected Nucleotides forming a backbone
(Phosphodiester bonds)
4
Q
Secondary Structure of Nucleic Acids
A
How the nucleotide chain folds or pairs within itself or with another strand
- DNA: Double Helix anti-parallel strands
- RNA: Hairpin loop, Bulges, Internal loops, Multi strand regions
5
Q
Which Nucleobase conformation is favored & why
A
Anti-conformation due to less steric hindrance (repulsive forces)
6
Q
Base pair bonds
A
- C-G: 3 hydrogen bonds
- A-T: 2 hydrogen bonds
7
Q
Are base pairs exactly perpendicular?
A
No
6˚ inclination
8
Q
Deamination reactions (CAG)
A
- C = Uracil (A)
- A = Hypoxanthine (C)
- G = Xanthine (C)
9
Q
A-DNA
A
- Right Handed
- 2.6nm diameter
- 11 Bp/turn
- Anti conf.
10
Q
B-DNA
A
MOST COMMON
- Right handed
- 2.0nm diameter
- 10 Bp/turn
- Anti conf.
11
Q
Z-DNA
A
- Left handed
- 1.8nm diameter
- 12 Bp/turn
- Pyr: Anti , Pur: Syn
12
Q
Bonds linking sugar to phosphate
A
- Phosphoester bond (-diester in backbone)
- Phosphoanhydride bw phosphates when multiple (ATP, ADP,…)
13
Q
Tautomers
A
Isomers differing in position of Proton or Electron
- Oxo-Enol (pur)
- Amino-Imino (pyr)
14
Q
What are the physiologically preferred Tautomer forms of Bases
A
Oxo (=o) & Amino (NH2)
15
Q
Heterochromatin (+types)
A
Condensed, transcriptionally inactive
- Facultative: Can become Euchromatin
- Constitutive: Cannot serve as a template (permanently cond.)
16
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)
17
Q
Histones that form Histone Octamer
A
- H2A 2x (dimer)
- H2B 2x (dimer)
- H3 2x (tetramer w H4)
- H4 2x (tetramer w H3)
18
Q
H1 Histone protein
A
Linker protein, clamps nucleosome
(most positive charge)
19
Q
How can histones bind DNA?
A
Histones are positive due to Arginine & Lysine binding negative DNA due to phosphate groups
20
Q
Nucleosome
A
DNA wrapped twice around a histone octamer where minor groove faces histones
21
Q
Chromatosome
A
Nucleosome bound to a H1 histone protein and adjacent Linker DNA
22
Q
Non-Histone Proteins
A
- HMG (high mobility group)
- SMC (struct. maintenance of chromosome)
Bound to Linker DNA
23
Q
Condensin
A
Type of Structural maintenance of Chromosome (SMC) that compacts DNA by loop extrusion & stabilizes it (ATPase)
(Activated by mitotic CDK-MPF)
24
Q
Radial loop regions
A
- SAR (scaffold)
- MAR (matrix)
- CCCTC region binds CFTF protein which helps Radial loop formation
25
Radial loop & Chromosome Other names
- Radial loop: Extended Scaffold Associated form
- Chromatin: Condensed Scaffold Associated form
26
What phase do we usually find chromatin in?
Interphase
27
Superspirilization in Prokaryotes
- Positive Superspiralization (Overwound DNA, right)
- Negative Superspiralization (Underwound DNA, left)
28
Linking Number
Number that describes linking of 2 closed circular DNA
Twists + Writhes
29
Twists
Right-handed helical turn
30
Writhes
Suprahelical turn in Negative Left-handed orientation
(unwinding a twist leads to writhes)
31
Topoisomerases (+ active site)
Enzymes that relieve Torsional stress during DNA replication
Tyrosine active site
32
Type I Topoisomerase
- Acts on 1 strand only
- No energy
- Changes linking n. by 1
- Creates nick
- For both neg/pos. supercoiling
(only for negative supercoiling in Prokaryotes)
I, III
33
Type II Topoisomerase (& example)
- Acts on both strands
- Requires ATP
- Changes linking n. by 2
- For both neg/pos. supercoiling
- e.g DNA Gyrase (II), IV
34
Human Genome
All genetic information in a cell which includes Nuclear and Mitochondrial DNA
(apx. 3 Billion BP)
35
Length of the Human Genome
~2m
36
Mitochondrial Genome info & total genes
Circular DNA with No Histones, No Introns!
Typically 5-10 copies in each mit.
37 total genes, 13 protein coding.
22 tRNA
2 rRNA
(mRNA is Polycistronic)
37
Polycistronic
One mRNA can code for multiple proteins by default
(only in prokaryotes/mitochondria)
38
What does Mitochondrial DNA lack?
5' Cap
39
5' Cap
Modified Guanosine neuc. added to 5' end of mRNA in Eukaryotes, important in initiation of translation
40
What determines the complexity of the Human Genome?
The number of non-coding regions (introns)
41
Gene
Entire nucleic acid sequence that is necessary for the synth. of a functional gene product
42
What are Eukaryotic genes made up of?
- Promoter region
- Open Reading Frame (ORF)
43
Promoter region (+ parts)
Initiates Transcription & Binds RNA Polymerase
- Core promoter
- Proximal promoter
44
Core promoter
- Region initiating transcription
- Initiator element (Inr) with Start codon
- DPE (downs. prom) & MTE (motif ten e) for pre-initiation complex
- TATA Box
- B-recognition element (BRE)
45
Downstream Promoter Element (DPE) function
Binds Factor II D which helps recruit RNA Polymerase
46
TATA Box
- Rich in Thymine and Adenine
- Defines direction of Transcription
- Recognized by TBP, TFIID
- (-25)
47
B-recognition element (BRE)
- Upstream of TATA box
- Binds Factor II B, to start transcription (starting point)
48
Initiator element (Inr)
- Recognized by TFIID
- Near transcription site
- Can sub. TATA
49
Proximal/Regulatory Promoter
- Upstream of Core Promoter
- CAAT Box
- GC Box
50
CAAT Box & GC Box
- Binds regulatory proteins or transcription factors (enhancers)
- (-80) CAAT
- (-100) GC (can be methylated)
51
Open Reading Frame (ORF)
Region of the gene that is transcribed to an RNA molecule from START to STOP codons (exons + introns)
52
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 to interact with RNA polymerase
53
Mechanisms of Enhancer Regulation
1) Rigid Model (Enhanceosome): IFN-B
2) Flexible Model (Billboard): steroid h exp.
3) Collective Model
54
Hormone Responsive Elements
Type of enhancer which recruits hormone receptors on:
- Palindromic seq. (homodimers)
- Direct repeats (heterodimers)
- Half-repeat (monomers)
55
3 Types of Exons
1) Initiator Exon (splice site + start)
2) Internal Exon (2x splice site)
3) Terminal Exon (splice site + stop)
56
Longest Gene & Most exons
- Longest: Dystrophin, 2.3 MB, ~80 exons
- Exons: TITIN gene, 312 exons
57
Alternative Splicing
Process that allows single gene to code for multiple proteins
So exon can be intron of other protein (vice versa)
58
Insulators (+ types)
Regulatory gene sequences binding insulator proteins
- Barrier insulators
- Enhancer insulators
59
Barrier Insulators
Sequence before a gene preventing heterochromatization, allowing transcription
60
Enhancer Insulators
Sequences between enhancers and promoters to stop DNA loop formation
(No interaction of promoter & e/s)
61
Psudogenes
DNA sequences resembling a functional gene but have MUTATIONS that prevent proper expression
(Gene-Like structures)
62
Pseudogene functions
- Can code for Pgk2
- Used as template for transcription (non-coding)
- 3D chromatin interaction (stabilize DNA)
- Reactivation, gene conversion (rare)
63
Direct Pseudogene formation
- Gene mutation (Point)
- Non-processed pseudogene (from duplication)
e.g L-gulonolactone oxidase showing humans were once able to produce VitC (gene fossil)
64
Indirect Pseudogene formation
Portion of the mRNA or RNA transcript of a gene is spontaneously reverse transcribed back into DNA and inserted
into chromosomal DNA (processed pseudogene)
65
Transposons
Segments of DNA that can move around to different positions in the genome of a single cell
(jumping genes)
66
Class II Transposon
- DNA Transposon
- 1 enzyme
- Sequence cut and pasted to new place
- Transposase enzyme used
67
Class I Transposon
- Retrotransposon
- 3 enzymes
- Copy and paste but RNA (RNA poly)
- RNA then back to DNA (reverse transcriptase) then integrated (Integrase)
So here sequence is duplicated
68
Types of Interspersed Repeats
- LINEs
- SINEs
- LTR Elements
69
LINEs
Long Interspersed Nuclear Element
- Retrotransposon
- Codes for ORF1 (p40 mRNA BP) & ORF2 (endonucleases & rev. transc.)
e.g. LINE sequence insertion is one of the known mutations in Hemophilia
70
SINEs
Short Interspersed Nuclear Element
- Parasite transposed element using LINE elements to multiply (ORF1/2 & reverse transcriptase and endonuclease)
71
LTR Elements
Long Terminal Repeat Elements
- Retrotransposons
- Found at end of retrovirus genome
- For integration of Virus into host DNA
72
What virus is integrated in Human genome
HERV
Human Endogen Retrovirus
8% integration
- Dangerous cause some viruses shown to activate HERVs causing autoimmune reaction (Mult.scl)
73
Tandem Repeats
Repeat sequences one after the other with no breaks or bases in bw
Formed by DNA slippage
74
Types of Tandem Repeats (VNTRs)
- Microsatellite (1-9 bp)
- Minisatellite (10-100 bp)
- Macrosatellite (100+ bp)
75
Tandem repeats Disease example
Fragile X Syndrome
Mutations in FMR1 Gene due to Tandem repeats
(CGG repeat)
76
General Replication Mech (semiconservative)
1) Helicase unwinds dsDNA using ATP at replication origins (AT-rich)
2) RNA polymerase called Primase forms a short 10-15 nucleotide primer
3) DNA dependent DNA polymerase elongates primer forming a mixed primer
4) For lagging strand, Okazaki fragments form since Primase makes new primer to keep 5' to 3' repl.
77
Type I DNA Dependent DNA Polymerase (Prokaryotes)
- Synthesis, 3' & 5' Exonuclease activity
- Particularly works on Lagging strand, lower processivity
- Role in repair
78
Type II DNA Dependent DNA Polymerase (Prokaryotes)
- DNA Repair (induced by damage)
- Synth & 3' Exonuclease only
- Not required for DNA replication
79
Type III DNA Dependent DNA Polymerase (Prokaryotes)
- Replication (higher processivity)
- α, ε, and θ Subunits for synth.
- 2 B subunits for attachment to DNA
- Synth & 3' Exonuclease only
80
OriC
The 1 single defined replication origin in Prokaryotes
- Rich in Adenine & Thymine nucleotides (since only 2H bonds, easier to separate)
81
DNA separation in Prokaryotes
- dnaA protein recognize OriC and bind dnaA box
- dnaB & dnaC form Helicase-like complex all together
82
Primosome
Group of proteins that help initiate DNA replication by setting up proper conditions needed for synthesis
83
Parts of the Primosome
- HD-proteins: Keep DNA single stranded
- N-proteins: attached to dnaB & dnaC
- Primase / dnaG: Synth. RNA primer
84
Replisome
Combination of Primosome and DNA polymerase III
85
What does DNA ligase use for energy in Prokaryotes?
NAD+
(Eukaryotic uses ATP)
86
What happens in G1 to unwind?
(Eukaryotes)
1) ORC hexamer binds to starting point on DNA
2) CdC6 (2) & Cdt1 (2) proteins form complex similar to dnaB/C (for helicase)
3) Mcm (2) also binds forming pre-initiation complex, active helicase
(cdc6 & cdt1 keep mcm inactive for now)
87
Cyclins
Family of regulatory proteins
- Can switch on specific CDKs
- CDKs phosphorylate proteins based on which phase its in
88
What happens at start in S phase?
1) Cdc6 phosphorylated by CDKs
2) Mcm not inhibited anymore by cdc6
3) Helicase activity
4) Topoisomerase I associates parent DNA ahead to relieve torsional stress
5) ORC phosphorylated and inhibited to prevent re-initiation
89
α - DNA Polymerase
- Forms tetramer with Primase
- Only synthesizes initial DNA segment after RNA primer
- Extends RNA primer by 25-ish nucleotides (mixed RNA-DNA primer)
- No 3' exonuclease, makes mistakes
90
β - DNA Polymerase
DNA Repair
(similar to DNA polymerase I in prokaryotes)
91
γ - DNA Polymerase
DNA synthesis in the Mitochondria
92
δ - DNA Polymerase
DNA synthesis on Lagging strand
93
ε - DNA Polymerase
DNA synthesis & repair on Leading strand
94
PCNA (proliferating cell nuclear antigen)
- Sliding clamp forms complex with δ/ε
- Ensures that δ and ε DNA polymerases dont dissociate from template
- Increases processivity of the polymerase
(homotrimeric)
95
RFC (replication factor C)
- Clamp Loader
- Inserts and opens PCNA ring to encircle region of DNA synthesized by polymerase alpha
(pentameric)
96
What happens on Okazaki fragemnts synth. (eukaryotes)
1) RPA (rep. protein A) binds ssDNA to stabilize it
2) Pol δ then binds and removes RPA and synthesizes strand until RNA-DNA primer
3) Rnase-H removes most of RNA primer, FEN1 removes RNA-DNA junction (5' exonuc.)
4) Pol δ fills gap, DNA ligase seals it
97
Telomerase
Enzyme that adds telomeric repeat sequences (TTAAGG) to 3' ends of each chromosome to compensate for shortening due to RNA primer removal on lagging strand
(reverse transcriptase)
98
Hayflick limit
Limit of cell division before the telomere is too short
(Ageing)
99
What stabilizes the T-loop (telomere)
Shelterin complexes
- TRF 1/2 (inhibit telomerase)
- TIN2
- TIPP1 (activates telomerase)
- POT1
100
POT1 and TRF2 significance
(+ what activates TRF2)
Shelterin proteins that inhibit ATM and ATR which are kinases that recognize telomere as damage and initiate repair
(TRF2 activated by RAP1)
101
p53
Tumor supressor protein
- Checks DNA during G1 before replication
- All corrected before S phase
102
Common types of DNA damage
- Thymine dimers
- Modification of bases by exo/endogenous agents (alkylation/intercalators)
- Loss of Purine base
- Deamination
103
DNA intercalators
Modify structure of the double helix by fitting bw bases/strands causing breakage and stoppage of replication
104
What types of DNA damage happen spontaneously?
- Loss of a Purine base
- Deamination
105
Direct Repair
Damage is identified and corrected
(present in prokaryotes to repair Thymine dimers using photolyase)
106
Base Excision repair
Damaged base is removed and missing part of the chain is resythesized
(Deamination & loss of purine base repair)
107
Nucleotide Excision repair
Entire DNA segment with the false conformation is removed , then resynthesized
(Repair of thymine dimers)
108
Mutation
Permanent alteration in DNA (<1%)
109
Types of chromosome mutations
- Deletion, Insertion, Repeats
- Inversion
- Translocation
110
Polymorphism
Genetic variation that is present in population with high allele freq. (>1%)
111
DNA Deamination
Loss of an amino group from Bases of DNA, generating a "foreign" base either by Ionization or spontaneously
1) Oxidative deamination (ROS) or Spontaneous
2) Keto group replaces Amino
- Cytosine most common
112
Repair of DNA Deamination
Base Excision Repair
1) Glycosidase enzyme removes deaminated base (AP site)
2) AP endonuclease removes deoxyribose-phosphate
3) DNA polymerase I/B resynth.
4) DNA ligase adds ester bond to close break
113
Thymine dimers
2 Thymines break their double bonds with Adenine and form 2 single bonds with each other
(also C-C or C-T)
114
What causes Thymine dimers
UV light
by electron excitation
115
Why is Thymine dimer bad
2 Thymines will be read as 1, so only 1 adenine is added to the strand, so a deletion basically
(replication and transcription inhibited)
116
Repair of Thymine Dimers
Nucleotide Excision Pair
1) Specific endonuclease used to remove entire segment
2) DNA polymerase I/B resynth.
3) DNA ligase adds ester bond
117
Difference between base and nucleotide excision pair
Glycosidase is used first in a base excision
in Nucleotide excision, we use a special endonuclease only which preforms the 2 steps in one go
118
Repair of Thymine Dimers in Prokaryotes
Direct repair
Photolyase enzyme which can reverse dimerization
Activated by UV light
119
DNA mismatch (& frequent cause)
DNA damage caused by incorrect pairing of 2 bases on double helix
(frequently due to oxo-enol tautomerism)
120
DNA mismatch example (guanine)
Normally favorable in Oxo form = oxo-guanine (binds C)
- If tautomer enol-guanine, it binds T instead
- We get G-T, forming a kink
121
Repair of DNA mismatch, how do we know which base is incorrect?
During synthesis, the template/original strand is methylated and the synthesized isn’t methylated for a while
122
Repair of DNA mismatch
1) MutS enzyme recognizes the mismatch
2) MutH endonuclease attaches to methylated side at wrong base (inactive)
3) MutL activates MutH to cut region of unmethylated DNA, & UvrD helicase unwinds
4) Exonuclease (RecJ, ExoVII, Exo1) breaks strand from hemi-methylated region until mismatch
5) DNA polymerase III and Ligase (not pol1 cause slow, we need a strand)
(In euk. MutS/L = MSH/MLH)
123
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
124
Sense, Synonymous, Silent mutation
Nothing happens to the amino acid sequence
(due to redundancy)
125
Missense Mutation
Change of one of amino acid
126
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
127
Nonsense mutation
Amino acid codon is replaced by a stop codon
Truncated protein (shorter than it should be)
128
Frameshift
When a base is inserted or deleted from the coding sequence causing shift.
(can also cause a nonsense mutation)
129
What could happen if there is a Point mutation/SNP in a non-coding region?
- Regulation alteration or gene expression change
- Or simply nothing :)
130
Heritability
- H=1 differences in traits are purely genetic
- H=0 diference in traits is only caused by environmental factors
131
Diseases caused by Trinucleotide repeats
- Fragile X Syndrome (FMR1, CCG)
- Huntington disease (HTT, CAG)
- Myotonic Dystrophy (DMPK, CTG)
132
Diseases caused by Point Mutations
- Sickle Cell Anemia
- PKU
133
Diseases caused by SNP
Cystic Fibrosis
134
Huntington Disease
CAG repeat (poly-glutamine in protein)
Proteases cant degrade proteases
(neurodegenerative)
Autosomal Dominant
135
Sickle Cell Anaemia
Point Mutation of Hb B-gene
Glu to Val change
Altered surface molecules
Recessive allele
136
Phenylketonuria
Mutation of Phenylalanine Hydroxylase
Cofactor Deficiency (BH4)
137
Cystic Fibrosis
CFTR protein mutation
Nonsense Mutation
Autosomal Recessive
(Genetherapy, adenovirus containing CFTR gene)
138
Methods of Identifying Genetic Factors
- Genome wide association study (GWAS)
- Candidate Gene Analysis
- Case-control Study
- Transmission disequilibrium test
139
Genome Wide association study (GWAS)
- Polymorphisms are in the whole genome
- Compare genes of affected with healthy
- No hypothesis needed
- Stats. analysis for correction of multiple tests
140
Candidate Gene Analysis
- Hypothesis set before analysis
- Selected candidate genes analyzed
- Sometimes important targets can be left out
Further studied in Case control & Transmission Disequilibrium
141
Case Control Study
Allele or Genotype frequencies of Case and Control groups compared to check for difference.
(GWAS & Candidate used)
142
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
143
What does PCR stand for
Polymerase Chain Reaction
144
PCR vs Normal replication
PCR only a small part of the genome is replicated, while in normal the whole genome is replicated
145
How do we separate strands in PCR?
High temperature only, no proteins needed
(denaturation step)
146
How many cycles of replication in PCR?
35 - 40 semi-conservative replication cycles in a Thermocycler
(each cycle doubles quantity)
147
3 steps of PCR
1) Denaturation
2) Annealing
3) Elongation
148
PCR Denaturation
- 90 - 95˚C
- Separation of Strands
- Only hydrogen bonds break
149
PCR Annealing
- Cooled to 50-72˚C
- 25 - 30 seconds
- Allows Primers to Anneal/Bind
150
Primers in PCR
Are DNA!!!
ssDNA (16-30) synth. in test tubes and needed because DNA polymerase can only elongate
151
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-specific binding of primer
152
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
153
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.
(right length increases exponentially)
154
Components of PCR reaction mixture
- DNA template
- ssDNA oligonucleotide primers
- dNTPs (ACTG)
- Taq DNA polymerase (therm.stab)
- Buffer (for optimal env. + ions like Mg)
155
What do we use to see PCR results?
Agarose Gel Electrophoresis & DNA ladder to compare.
+ Intercalator dye (Ethidium Bromide) highly toxic!
156
How are results viewed on Real-time PCR
Fluorescent dye used, as the DNA increases the fluorescence also increases which is detected by a machine
157
Applications of Real-time PCR
- Gene expression analysis (mRNA)
- Gene dosage measurement
- SNP genotyping
- Novel polymorphism identification
158
Novel Polymorphism (+ how identified using PCR)
Genetic variation in DNA that has not been previously characterized or documented
- Real-time PCR used with HRM (high res. melting) with an intercalator dye. If any abnormalities in melting time, it shows difference in bondings (AT, CG)
159
Normal person to person Genetic Variation
~0.1% (MAX 0.5%)
160
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
Tells you exact place of base on strand
161
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)
162
Primer Extension Method - Mini Sequencing
'Mini' because we need 1 single primer
Only elongate primer by 1 nucleotide using color labelled ddNTPs, added complementary next to primer & check fluorescence
163
Restriction Fragment Length Polymorphism (RFLP)
Changes a sequence difference to a length difference
e.g. C/T Polymorphism
- Type II restriction endonuclease needed (highly specific), e.g. Targets T so if present cuts then analyzed by Gel Electrophoresis
164
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 = no product
- 3' exo. would just cut the wrong base and keep going which defeats the whole purpose
= So we check is there a PCR product generated or no?
(can be Bidirectional where 2 different primer 3' are there and tests genotype)
165
Prokaryotic Transcription Strands
- Template strand (Antisense)
- Coding strand (Sense)
166
Can transcription start without a primer in Prokaryotes?
Yes, because RNA polymerase is used
167
Proofreading in RNA polymerase?
No, no 3' exonuclease activity as RNA copy leaves the nucleus so no need, wont be genetically inherited
(has substrate binding & catalytic sites)
168
How does RNA Polymerase elongate?
NTPs have a,B,y phosphates
1) a pi on 5' carbon of ribose heterolysis by RNA polym.
2) Pyrophosphate (PPi) detached
3) Pyrophosphatase breaks second macroergic anhydride bond
4) a-Pi binds 3' C of ribose already on chain
169
What is prokaryotic transcription unit split into?
- Promoter region (-10, -35)
- Transcribed region
170
2 forms of RNA polymerase
- Holoenzyme
- Apoenzyme
171
Holoenzyme RNA Polymerase
Core + σ subunit
5 subunits (2a, B, B', σ)
- Can initiate transcription due to σ subunit interaction with promoter
- Low affinity (1s) /searching
- σ70 is specific for -35/-10
172
Apoenzyme RNA polymerase
4 subunits (2a, B, B')
Can only elongate the RNA chain
High affinity (60min) /holding
173
Is helicase used in Prokaryotic transcription
No
Holoenzyme RNA Polymerase can unwind helix, one turn at a time
174
Inhibitor of RNA polymerase in Prokaryotes
Natural Rifamycin or Rifampicin
- Binds to B subunit and prevents initiation
- Not absorbed in Gut so kills gut bacteria
175
Termination of Transcription types in Prokaryotes (2)
- Rho independent termination
- Rho dependent termination
176
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 by A-rich on template
4) RNA strand pulled out of pol. due to weak U-A H-bonds.
177
Rho dependent Termination
1) Rho (ρ) factor Helicase follows RNA polymerase
2) Uses ATP with every step
3) RNA poly. slows down, Rho (ρ) factor catches up
4) Rho (ρ) factor pulls RNA strand out, because no U rich sequence after
(Rut element, CA-rich, attracts Rho)
178
Post-transcriptional RNA modifications in prokaryotic cells
(+ example)
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 by tRNA nucleotidyltransferase
179
Regulation of Transcription in Prokaryotes
- Strong/Weak Bacterial promoters
- σ subunit replacement for new promoter
- Activator/Repressor reg. protein on Operator regions
180
Operon
One transcription unit that can code for more than one protein, which codes for the polycistronic mRNA
(Only Prokaryotes)
181
Ribosome binding sites (RBSs)
Located upstream of Start codon
1st cistron RBS in 5'UTR, none in 3'
(Shine-Delgarno Sequences)
In Eukaryotes it just binds 5’, no defined region
182
Lac Operon E.Coli
- Z,Y,A protein coding sequences with same promoter
- So polycistronic mRNA with 3 cistrons
- Codes for 3 enzymes:
1) B-Galactosidase
2) Galactoside Permease
3) Galactoside transacetylase
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Regulation of Lac Operon E.Coli
- Depends on Glucose presence (1' energy)
- Lac1 gene codes for Lac Repressor
- Lac repressor only removed if Lactose present as Ligand
- cAMP-activated CRP protein enhances RNA poly. binding (low glucose, high cAMP)
= Low Glucose + High Lactose needed
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Name of immature mRNA
premRNA or hnmRNA
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RNA Polymerase Eukaryotes
Type II
premRNA to mRNAs
- Can not initiate transcription
- 12 subunits
- Similar to apoenzyme
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a-amanitin
Death cap mushroom poison that inhibits transcription by inhibiting RNA polymerase
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Initiation of Transcription in Eukaryotes
Pre-initiation complex
1) TFIID TBP binds TATA aided by TFIIA
2) TFIIB binds BRE joining TFIID recruiting TFIIF bringing RNA Polymerase II
3) TFIIH docks at TFIIE & unwinds double helix at INR using ATP
- The TFs dissociate, but TFIID remains at TATA
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Pre-initiation complex Eukaryotes
- TFII A/B/D/E/F/H
- RNA Polymerase II
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Termiantion of Transcription in Eukaryotes
1) CPSF and CstF follow RNA polymerase II through process
2) CPSF detects stop codon (Poly-adenylation of transcript)
3) CPSF binds with high affinity to sequence and detaches from polymerase with help of CstF to cut.
4) Poly-Adenylate polymerase adds 150-200 adenines on 3' end
5) PAB (polyadenylate bind.) proteins recruited to cover tail
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CPSF & CstF names
- CPSF: Cleavage and Polyadenylation Specificity Factor
- CstF: Cleavage Stimulation Factor
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Exon def
Part of the gene that remains in the mRNA after maturation
(not protein coding since we have 5'/3' UTR)
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mRNA maturation parts
- 5' Cap
- Splicing of introns
- PolyA tail
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5' cap formation & bond
1) 1st nucleotide is usually ATP, RNA terminal phosphatase trims 1 phosphate
2) GTP added to ATP by Guanylyl transferase enzyme, PPi released, 5'-5' triphosphate linkage
3) Methylated by methyltransferase enzyme (SAM donor)
= 7-methylguanosine
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5' cap function
- Maturation signal
- Protective measure (shields from 5' exonuc.) = same for poly-A but not safe from 3' so the longer the better
- Initiation of Translation (elF4E)
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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
3) Leaves exon-junction complex to show where intron was spliced
(alcoholysis not hydrolysis)
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snRNPs in Splicing
- U1: 5' splice site
- U2: Branching site (adenine)
- U5: 3' splice site
= ALL FORM SPLICOSOME
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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)
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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 BPs to direct mRNA to nuclear pore & push it out
3) 5' Cap binding complex (CBC) switched to Cytosolic cap binding proteins (eIF4E) after exiting
4) Loop forms, Poly-A tail touches 5' Cap
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What regulates Initiation of Euk. Transcription
Mediator Complex
- Enhance or block transcription initiation by receiving signals from activators or repressors to RNA polymerase II
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Spacer DNA
- Long undefined sequences of DNA (100s/1000s nucleotides)
- Between the regulatory sequences
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What is the main way Euk. Transcription is Regulated
Packing/Unpacking of heterochromatin and Euchromatin
since in packed DNA TATA box is not available
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What causes unpacking of DNA to euchromatin
Prime activator proteins which recruit co-activators when bound to enhancer sequence like Histone acetyl-transferases
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2 Pathways after Prime activator
- Chromatin Remodeling Complex (loosens nucleosomes)
OR
- Histone Modifying Enzyme (acetylation to reduce + charge)
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What happens when DNA unfolds and Core promoter is seen to help unfold
1) Activator protein binds to enhancer sequence
2) Activator protein recruits Histone acetyltransferase acetylating lysine residues
3) Histone Kinase also recruited phosphorylating (-) serine on histones for more repulsion
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Histone Code
Pattern of modifications happening to loosen DNA by histone kinase and histone acetyltransferase
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What does Histone code recruit and what does it do
Chromatin Remodeling complex
(different than co-activator CRC)
- Stabilizes the less condensed chromatin, keeping the DNA accessible.
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TFIID role in Histone Code
TFIID also binds the histone code as well as core promoter
So it can engage the initiation of Transcription
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Ways repressor proteins can act
- Block binding of Activator
- Binds to silencer sequence and competes with bound activator-enhancer
- Repressor and activator compete for mediator complex
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Co-repressors
- Chromatin remodeling complex
- Histone deacetylase & Histone methyltransferase
(both increase condensation)
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Specific Transcription factors
- Help regulate gene expression by binding to specific DNA sequences called Response Elements
- Usually Dimeric (homo/hetero)
- Can be part of repressor or activator complexes
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Regulation of Specific Transcription factors (reg. examples)
- Protein Synthesis (under transcriptional control itself)
- Ligand binding (Nuclear-R/orphan)
- Covalent Mod. (undoable)
- Dimerization
- Unmasking (removing inh.)
- Stimulation of Nuclear Entry
- Release from membrane (because they are anchored)
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DNA-Bending protein
Not repressor or activator, but helps both by bringing regulatory sequences close to the core promoter
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DNA recombinant technology
Process of combining DNA from different sources to create a new DNA sequence (recombinant DNA)
e.g. Insulin
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Plasmid
- Circular double-stranded DNA
- Replicate independently from genome
- 1-2 protein coding genes usually for protection against toxin/antibiotic
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Vectors
DNA molecules which will carry foreign DNA insert into host cells
(Plasmids/Bacteriophages)
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Inserts
- Target DNA which will be inserted into the vector
- Contains a specific gene which codes for the protein we wanna produce
- Can only contain exons since bacteria dont splice
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Restriction Endonucleases
- Bacterial enzymes (like scissors)
- Cut DNA at specific recognition sites
- Each Restriction Endonuclease is specific to a particular recog. site
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Sticky end
- Restriction Endonucleases cuts DNA leaving single-stranded overhangs
- If 2 sticky ends are complementary they can form H-bonds in a NaCl solution
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Blunt end
Restriction Endonucleases cut straight through DNA with no overhangs
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Why do we want sticky ends?
With blunt ends, Vector can just re-ligate without the insert
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How to prepare for PCR
- 2 Primers designed
- 5' ends have recognition sites which are not complementary but for specific Restriction Endonucleases
- PCR product = insert
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Transferring the Insert into the Vector
1) Plasmid vector also modified to have 2 recognition sites for specific Restriction Endonucleases
2) Insert and Vector have sticky ends complementary to each-other
3) H-bonds form and any breaks joined by DNA ligase
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2 Types of Vectors
- Expression Vector (plasmid)
- Reporter Vector
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What makes up an Expression Vector?
- Ori
- Promoter
- Selective Marker Gene
- Gene of other protein/peptide (tag)
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Expression Vector Promoter
- Active: Strong promoter
- Constitutive: can be activated
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Expression Vector Selective Marker Gene
e.g. Antibiotic resistance gene
Important to figure out which ones would survive in presence of that specific antibiotic
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Expression Vector Gene of other protein/peptide
- Tag (glutathione transferase) sequence or fusion protein
- e.g. 6-histidine tag (in lab)
- For purification of the produced protein by Affinity Chromatography
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Transformation of Vector
- Introduction of recombinant DNA into host cells (we use bacteria for insulin)
- Bacteria uses machinery to make protein the gene codes for (insulin)
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How do we make sure the bacteria have taken up the Plasmid vector?
By having an antibiotic resistance gene and having the bacteria in presence of that AB, so only ones that survive took it in
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How to make bacteria take up the Plasmid Vector?
- Chemical via heat shock
- Electric
Pores form allowing DNA to enter
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Purification of the Protein
1) Tag sequence fused to target protein
2) Mixture poured into column and only tagged proteins stick to ligands
3) Everything else is washed away
4) To get protein out we change pH or salt conc. in elution to let go of the Ligand
5) Protein collected in fractions and purity/conc. is assessed
(Affinity Chromatography)
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Reporter Vector
- Designed to monitor/measure the activity or expression of a regulatory gene (promoter) which will be our insert
- Reporter gene (luciferase) used which creates a signal (light) when expressed so more light means stronger promoter
