1
Q
Beta Turns
A
- 4 residues in a tight turn, h-bonding between 1st and 4th
- 2nd and 3rd residues usually proline or glycine
2
Q
Non-Coding DNA Types
A
intragenic DNA (introns) intergenic DNA others: tRNA, rRNA, miRNA
3
Q
Transcription Numbering
A
- RNA polymerase parks itself on the promoter region (< -1)
- RNA polymerase starts reading anti-sense starting from +1 onwards, from 3’ to 5’
4
Q
Solitary gene
A
- gene represented only once in genome
5
Q
Intrinsically Disordered Protein (IDP)
A
- usually has many charges, and less hydrophobic
- trigger demixing in cells, matching proteins that usually don’t interact
6
Q
Simple Transcription Unit
A
- only one possible gene product, no alternative splicing
7
Q
Earmarking
A
- adding tag to polypeptide to indicate where it’s meant to go in the cell
8
Q
Relative Mass of Cell Components
A
Largest Mass
- nucleus (including transcription factors, DNA)
- chloroplasts, mitochondria, lysosomes, peroxisomes
- membrane, ER, polyribosomes
- ribosome subunits, small polyribosomes
- citosol
Smallest Mass
9
Q
SV40 Virus
A
- model organism for studying DNA replication
- short circular double-stranded DNA
- only affects eukaryotes, namely monkeys/humans
- replicates very fast using host cell machinery, with the exception of Large-T Antigen
10
Q
Pol delta
A
DNA Polymerase delta
- completes Okazaki fragments
11
Q
Alpha Helices
A
H-bond happening in a unidirectional pattern between the amino and carboxyl groups 3.6 residues away on avg.
12
Q
Popular Protein Affinities
A
Protein tag: GST (glutathione transferase)
Substrate: Glutathione, bound to agarose
Protein tag: Containing many His
Substrate: Nickel
13
Q
Plasmid Cloning Vector
A
- ORI
- drug resistance gene (ex: ampr)
- polylinker
14
Q
Leading strand
A
- unwound from 5’ to 3’, and synthesized from 3’ to 5’, so synthesis can happen continuously
15
Q
Protein Domain Types
A
Globular - well folded and soluble Fibrous - not well folded, but sturdy - not well soluble - many parallel structures Integral - connects to membrane Intrinsically Disordered Protein (IDP) - "other"
16
Q
Protein Crystallography
A
- condense proteins into crystal
- shine high powered laser on it
- analyze refraction patterns
- used to infer protein shape
17
Q
Insulator
A
- forms barriers between genes
18
Q
DNA Cloning using Plasmids
A
- with plasmid cloning vector:
- T4 DNA Ligase, very efficient bacteriophage ligase
- CaCl2 and heat pulse to insert plasmid into bacterial DNA
- ampicillin plates to select for target
19
Q
PCNA
A
- homotrimeric
- prevents complex from disassociating from the strand, wrapped around strand
20
Q
EF hand motif
A
- binds calcium ions
- acts like a hinge
- helix-loop-helix pattern
21
Q
2D Electrophoresis
A
- seperate protein by both mass and charge
Process:
- using ampholytes, create pH gradient in gel
- apply isoelectric focusing
- run gel on SDS-PAGE
22
Q
Restriction Endonucleases
A
- cuts dsDNA at a specific palindromic sequence, in a symmetrical manner, sticky or blunt
- methylated DNA is protected
23
Q
UPR
A
- Unfolded protein response
- leads to the production of more chaperone proteins
- in extreme case, leads to cell death
24
Q
Epitope
A
Location on antigen which antibody binds to
25
Eukaryote Transcript Parts
- Poly(A) tail, starting at end of last exon
- G cap, ending at start of first exon
- to prevent degredation
- usually contains introns which are spliced out
- between cap and tail, contains UTR
26
CRM
- cis-regulated module
- non-translated region ~100-1000bp upstream acts on which proteins that adjust expression of gene bind to
- can act as enhancers, silencers, or insulators
27
Bacteria Expression Vectors
- used to over-express proteins of interest using inducible gene expression
- engineer plasmid with gene of interest downstream of promoter (ex: lac, to trigger with lactose)
28
cDNA Libraries Purpose
- goal is to represent mRNA in a given sample
29
Taq
- DNA Polymerase
- very from organism that lives in hot springs
- thermostable
30
Drosophila Hsp90 Experiment
- Hsp90 was knocked out by half in flies
| - genetic variants became much more apparent, including cancers, discolourations
31
Phosphorylation
- transfer of a phosphate group to a phosphoacceptor from the gamma phosphate of a NTP
32
Zinc Finger
- motif that binds a zinc ion using histidine and cysteine residues
33
Protein Phosphatase
- removes phosphate from a particular substrate
34
Transient Transfection
- insert plasmid that contain viral ORI into cells
35
PCR Ingredients
- DNA template (sweet spot is ~1000 bp, can go up to 20kbp)
- two different primers (DNA, oligonucleotides ~20 bp), one for each end of the fragment
- dNTPs
- Taq
36
LC-MS-MS
- tendem mass spectrometry following liquid chromatography
Process:
- trypsination of protein sample, usually quite pure
- feed into liquid chromatography column
- fractions go into an MS, get analysed for molecular weight
- MS fragments fractions further, and infers AA sequence using database of fingerprints
37
Dissassociation Constant
- Kd
| - ratio of [A][B]/[AB], if AB is a protein complex of A and B
38
Amyloid plaques
- aggregates of misfolded proteins, causing cell death
| - associated with Alzheimer Disease, Parkinson’s, ALS
39
Hsp ATP cycle
ATP bound - open, exposing hydrophobic domain
ADP bound - closed, protein allowed to fold
- multiple cycles are sometimes needed to fully fold protein
40
Dideoxy Chain-Termination Method and Ingredients
- automated DNA sequencing technique
- single tube reaction:
- DNA Polymerase
- Primer (oligonucleotide)
- DNA template (usually amplified through PCR)
- dNTPs (100 mM)
- ddNTPs (1mM of each ATCG, dyed)
41
PCR Process
In a thermocycler:
- ~95C - denature DNA (almost 100%)
- ~60C - anneal primers
- primers are smaller and higher concentration, so they renature first
- ~72C - DNA extension (Taq ideal temperature)
Repeat 20-40 times to generate around a billion fragments
~4$ per run
42
Phosphorylation motif
- motif recognized by kinase
| - contains free OH group, the phosphoacceptor
43
Ubiquitination
- attach ubiquitin to target
```
Pathway:
E1 ubiquitin activating enzyme
- uses ATP, ligates itself to ubiquitin (thiolester bond)
E2 ubiquitin conjugating enzyme
- Ub transfered from E1 to E2
E3 ubiquitin ligase
- depends on target
- adds Ub to target
```
44
Replication fork
- aka growing fork
| - formed at point where DNA duplex is currently being unwound
45
ddNTP
- dideoxyribonucleoside triphosphate
| - chain terminator, cannot be used to elongate chain further
46
Initialization Complex for RNA Transcription
RNA polymerase
General transcription factors
Mediator complex
47
Cyclin Dependent Protein Kinases
- needed to cycle through cell stages
- uses cyclin to confer substrate specificity
- cyclins are polyubiquinated to make way for next ones
48
Gene
DNA necessary for synthesis of a functional product (polypeptide or RNA)
- usually entire transcription unit (including coding and regulatory DNA)
49
GTP Binding Proteins
- GTPase proteins use GTP as a switch
- GTPase Activating Proteins (GAP) turn protein off by hydrolysing phosphate
- Guanine Exchange Factor (GEF) turn protein on by exchanging GDP for a GTP
- some cancers are caused by defective a GAP protein
50
Gap repair
- Ribonuclease H and FEN 1 remove RNA
- Pol delta replaces RNA with DNA
- DNA Ligase ligates them
51
Western Blot
aka Immunoblotting
- identify protein of interest among other proteins in a gel
- apply primary and then hybridize secondary antibodies, the second usually chromogenic
- signal is proportional to amount of protein
52
Polyclonal Antibodies
- antibodies which are produced from different cell lineages, targeting the same protein, but at different epitopes
- produced by injecting animal with antibody, collecting serum and isolating antibodies that target protein
- contrasted to monoclonal antibodies, which target the same epitope
53
Immunostaining Proteins in situ
- using immunostaining (same technique as for Western blots) in order to visualize protein as it looks inside a cell
Process:
- fix cell to a microscope slide
- make membrane permeable using detergent
- incubate with primary antibody, then secondary fluorogenic antibody
- expose to UV light and view
54
Promoter
- region of DNA upstream of gene acting as on-off switch
| - not translated
55
Leading strand synthesis complex
Pol epsilon
RFC
PCNA
56
Ion Exchange Chromatography
- differentiate proteins based on charge (pI)
- used charged beads
- elute with competing ions, ex NaCl
57
Genome
- an organism’s entire hereditary data
- DNA + some viruses
- Eukaryoatic genome made up of coding islands and non-coding open oceans
- Human genome: ~3.3 Gbp
- genome not good predictor of organism complexity
58
GFP
- Green Fluorescent Protein
- beta-barrel structure, with an oxygen binding domain inside the barrel
- shines green when flash blue light
59
Gene Families
- a group of genes with very similar sequences, typically in gene clusters, usually formed by duplication
- 25-50% of proteins in humans part of gene family
60
Adenine Forms
Nucleoside - Adenosine
Nucleotide - Adenylate
dATP - deoxyribose adenosine triphosphate
rATP ribose adenosine triphosphate
61
RNA Structures
```
Hairpin:
- small loop, secondary
Stemloop:
- large loop, secondary
Pseudoknot:
- 2 loops emanating from a 2 stems, tertiary
- part of function of telomerase
```
62
Secretory Pathway
- nascent polypeptides are earmarked for ER
| - ribosome is bound to ER, and protein is translated directly into ER lumen, and modified concurrently
63
Pol alpha
DNA Polymerase alpha
| - elongates primer on lagging strand
64
Immunoprecipitation (IP)
- analytical analog to affinity chromatography
| - uses affinity beads to a target protein, which sediment first when centrifuged
65
DNA Barcoding Sequence Candidate
- must lack introns, insertions, deletions, in order to easily be aligned with reference genomes
- one candidate is CO1 inside mitochondria genome (mtDNA)
66
DNA Helicase
- MCM in eukaryotes (minichromosome maintenance)
- Large-T Antigen in SV40
- unwinds DNA
- following ORC, unwind duplex DNA going in opposite directions from origin
- unwinding is usually limiting factor in DNA replication speed
67
Hsp70
- monomer molecular chaperone
| - most characterized Hsp molecule
68
Gene Cluster
- dense region of DNA containing genes of the same family
69
Beta Sheet
- interact laterally. Either parallel or antiparallel.
| - can be used to form beta-barrels and beta-propellers
70
RFC
- Replication Factor C
| - loads the DNA polymerase complex onto the template
71
Lagging strand
- unwound from 3’ to 5’, and synthesized from 5’ to 3’, so synthesis needs to happen discontinuously
72
Amino Acid
- has N-terminal and C-terminal
- forms polypeptides
- L (levo) stereoismer dominant in life
73
Different Strands wrt. Transcription
Sense (coding strand)
| Anti-Sense (template strand)
74
DNA Forms
B Form: Right-handed helix.
A Form: Right-handed helix. Wider and squatter. Deeper major grooves, shallower minor groove.
Z Form: Left-handed helix. Narrower and longer.
Often appears when alternating purine pyrimidine in regular fashion.
- In vitro, In low-humidity, can convert B-DNA to A-DNA.
- In vivo, A-DNA exists when it’s hybrid DNA-RNA, or RNA-RNA db-helix.
75
Transfection
Introduce a gene into a eukaryotic cell
76
SSR Types
- minisatellie DNA (~14-100bp, 20-50 tandem repeat units)
- often in centromeres (center) and telomeres (ends)
- microsatellite DNA (~1-4bp, arrays up to 600bp of tandem repeats)
- sometimes in transcription units
- expansion related to neuromuscular diseases
77
Complex Transcription Unit
- multiple possible gene products using alternative splicing
| - a majority of genes in eukaryotic organisms
78
UTR
untranslated region (of DNA)
79
Trypsination
- apply Trypsin protease, which cuts polypeptide into many chains
- preparatory step to LC-MS-MS
80
Next Generation Sequence
- current state of the art is Illumina Solexa sequencing
- used for whole-genome sequencing
- takes about a day to run (1000-10000$ per genome)
81
Temperature of DNA Melting
- Tm
- function of GC-content, which makes Tm higher
- disassociation of DNA, measured by light absorption, follows hyperbolic function of temperature
- disassociation of RNA is linear
82
trans-regulatory module
regulates genes by producing proteins that in turn bind to the CRM
83
Pseudogenes
- inactive genes, remnants of once-duplicated genes
84
Co-Immunoprecipitation
- same as immunoprecipitation, but done more gently, in order to also analyze proteins that form complexes with target
85
Gel Filtration Chromatography
- size exclusion chromatography
- large proteins take the fast way down the beads, elute first
- use standard with known proteins to compare to proteins in fractions
86
Hsp90
- dimer molecular chaperone
- has ATP cycle
- more specific to signalling molecules than Hsp70
87
Heat Shock Proteins
- aka HSP
| - associated with cellular stress response
88
Affinity Chromatography
- use affinity beads in a chromatography column
| - elute with acidic buffer to make beads release protein
89
Ephrins
- ligand membrane protein used for cell-cell signaling
- interacts with its cognate ephrin receptor
- contains:
- transmembrane domain (hydrophobic)
- ephrin domain (hydrophilic)
- cytosolic tail (hydrophilic)
90
Supercoils
- coils of coils
- causes torsional stress
- forms on parental DNA duplex ahead of replication coil
- resolved with topoisomerases
91
ampr
- gene conferring ampicilin resistance
| - often used in engineered plasmids for selection
92
Origin of Replication
- target for ORC
- usually rich in AT
- not very conserved
- eukaryote chromosomes have many
93
ORC
- Origin Recognition Complex
- found in eukaryotes
- binds to origins of replication and associates with other proteins to load helicase
- six subunit protein
94
Protein Kinase
- phosphorylates a particular substrate
| - binds to a particular nucleotide (ex: ATP, GTP)
95
RPA
- Replication Protein A
- keeps single-stranded DNA from winding on itself, and in an optimal position for DNA polymerase which would slow down replication
- gripping strand using beta sheet finger-like projections
96
Plasmid
- circular extrachromosonal dsDNA, typical of bacteria & lower eukaryotes
- replication occurs before cell division, decoupled from DNA genome replication
97
SDS-PAGE
- separates proteins based on mass
- SDS - sodium dodecylsulfate, a detergent
- PAGE - polyacrylamide gel
Process
- boil proteins in SDS
- apply reducing agent to break disulfide bridges
- SDS binds to polypeptides, making them almost equally charged
- PAGE acts like a sieve, making large proteins run slower
- use standard
98
Prion Proteins
- aka PrP
- PrPc are in conformer (non pathogenic) state. alpha helix dominant
- PrPsc are in non-conformer state, and aggregate easily forming plaques. beta sheet dominant
- related to Creutzfeldt-Jakob, Kuru, BSE
99
Protein Tertiary Structures
- usually relies on hydrophobic interactions
- folding follows oil drop analogy; hydrophobic core and hydrophilic outside
- flexible structure; and same protein can fold in several different ways
100
BiP
- ER resident molecular chaperone
- when not active, is bound of Ire1, an ER membrane protein
- BiP disassociates when unfolded proteins are present, encouraging Ire1 to homodimerize
- homodimerized Ire1 triggers UPR that leads to the production of more chaperones
101
Popular Restriction Enzyme
EcoR1, sticky ends, from E-coli
102
IEF
- isoelectric focusing
- apply charge to gel with pH gradient
- molecules accumulate where charge is similar
103
Homolog Types
Ortholog - across species
| Paralog - in same species
104
Large-T Antigen
- hexomer
| - a highly efficient helicase protein, powered by ATP, found in SV40 genome
105
Disulfide Bridges
- cysteins are bound together in ER by PDI, Protein Disulfide Isomerase
- PDI is oxidized, or "reset" by Ero1
106
Dideoxy Chain-Termination Method Process
- denature and separate daughter strands by electrophoresis (with original DNA template eliminated)
- uses polyacrylamide gel as opposed to agarose
- more precise
107
26S Proteasome
- breaks polyubiquinated proteins down into amino acids, but recycles ubiquitins
- has 2 caps, 2 beta subunits, 2 alpha subunits in the middle, organized like a two-ended recycle bind
- depends on ATP
108
Motif
- chain of polypeptides usually no longer than 40 residues
| - confers a specific function to protein
109
Rate Zonal Centrifugation
- spin protein mixture in density gradient of sucrose (or other)
- proteins of similar mass (S units) collect in bands
110
General DNA Shape
- base pairs inside helix, backbone on outside
- major and minor grooves, where proteins can read DNA
- nucleotides bound to each-other by phosphodiester linkage
- backbone made of phosphate-pentose units
- 5’ end, free phosphate group bound to 5’ carbon of the terminal sugar
- 3’ end, free hydroxyl group bound to 3’ carbon of the terminal sugar
111
Differential Centrifugation
- run lysate sequentially in centrifuge
- in between spins, collect supernatant and pellet
- faster spinning puts lighter and lighter components in the pellet
112
Coiled coil
- motif containing alternating hydrophobic/hydrophilic residues
- example: leucine zipper
- polypeptide helices coiled around eachother
113
cDNA Library Creation Process
- isolate mRNA using T-oligo DNA primers tied to a matrix
- use reverse transcriptase to elongate DNA strand
- use RNase H to digest RNA, minus a few (primer) pieces
- use DNA polymerase to finalize second DNA strand
- PCR to amplify
114
Electropherogram
Used to validate base calling of Dideoxy Chain-Termination DNA sequencing
- some regions are hard to read (repeats)
- contamination might show overlapping peaks
115
Lagging strand synthesis complex
- Primase (specialized RNA polymerase) puts down a primer
- Pol alpha further elongates the primer with DNA
- Pol delta/RFC/PCNA complex replaces Pol alpha and primase and completes the Okazaki fragment
- at point of joining, ligated by DNA ligase
116
ORF
Open Reading Frame
| - coding region of DNA
117
PCR Goal
- polymerase chain reaction
| - amplifies a DNA fragment
118
Stable Transfection
- done when there’s a protocol available to fuse gene of interest into target cell genome
119
DNA Barcoding
- used to identity species uniquely, accurately and quickly
| - sensitive enough to differentate sequence between species, but not between mutations in the same species
120
Translational Fusion Proteins
- fusing proteins to other proteins of interest inside genome using recombinant DNA technology
- ex: tagging proteins such as GFP
- done by removing stop codon and replacing it with another protein
121
Michaelis Constant
- Km
- concentration of substrate when rate of reaction is half of Vmax
- always same for same enzyme-substrate relation
122
Pol epsilon
DNA Polymerase epsilon
| - synthesizes leading strand
123
Svedburg Units
- location of molecule in density gradient after spun in a centrifuge
- proportional to mas
124
Polylinker
- DNA region containing sites that are recognized by restriction endonucleases/enzymes
- can insert foreign DNA into it to form recombinant plasmid
125
SSR
- Simple Sequence Repeats
- sometimes used in DNA fingerprinting by looking at # of repeats (by PCR/electrophoresis)
- expansion can be caused by backwards slippage
126
Transcription Unit
- exons, control regions, and Poly(A)/G-cap of a particular gene
- control regions typically within 50kb upstream
127
NGS Sequencing Pipeline
- DNA isolation
- fragmentation (often mechanical sheering)
- library (that represents full genome)
- amplification
- sequencing
- dyed nucleotide terminators are reversible
- allow for further extension of same chain
- chains are annealed to surface using primer sequences
- fluids flow back and forth, laser reads the dye, the chain is elongated, etc
- assembly
128
Leucine Zipper
- interaction between two alpha helical regions, with a series of leucines on one side conferring hydrophobicity
- transcription factor
- dimer (homo or hetero dimers)
- binds DNA with recognition helices
129
Calmodulin
- multifunctional calcium modulated protein
| - binds calcium on an EF hand motif
130
DNA Libraries
mixture of all the DNA fragments from an entire genome inserted into recombinant plasmids
131
Polyubiquitination
- tag a protein for death
- ubiquitin proteins attached a Lysine residue on protein, in a chain
- monoubiquitin does not tag cell for death
132
Affinity Bead
- Agarose Bead -> Protein A -> Fc domain of IgG antibody
| - antibody used depends on which protein is targeted
133
Enzyme Structure and Catalysis
- binding site/pocket
- catalytic site/cleft
- lowers energy of transition state required for reaction to occur
134
Chaperonin
- type of chaperone
- very large protein
- highly conserved
- TriC in humans
- GroEL, GroES in bacteria
- has ATP cycles
135
Hemoglobin
- tetramer made up of 2 alpha units, 2 beta units
| - beta unit is highly conserved
BIOL 200
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