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BIOL 200 > Main > Flashcards

Main Flashcards

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

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2
Q

Non-Coding DNA Types

A 
intragenic DNA (introns)
intergenic DNA
others: tRNA, rRNA, miRNA
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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’

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4
Q

Solitary gene

A
  • gene represented only once in genome
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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

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6
Q

Simple Transcription Unit

A
  • only one possible gene product, no alternative splicing
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7
Q

Earmarking

A
  • adding tag to polypeptide to indicate where it’s meant to go in the cell
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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

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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
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10
Q

Pol delta

A

DNA Polymerase delta

- completes Okazaki fragments

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11
Q

Alpha Helices

A

H-bond happening in a unidirectional pattern between the amino and carboxyl groups 3.6 residues away on avg.

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12
Q

Popular Protein Affinities

A

Protein tag: GST (glutathione transferase)
Substrate: Glutathione, bound to agarose

Protein tag: Containing many His
Substrate: Nickel

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13
Q

Plasmid Cloning Vector

A
  • ORI
  • drug resistance gene (ex: ampr)
  • polylinker
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14
Q

Leading strand

A
  • unwound from 5’ to 3’, and synthesized from 3’ to 5’, so synthesis can happen continuously
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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"
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16
Q

Protein Crystallography

A
  • condense proteins into crystal
  • shine high powered laser on it
  • analyze refraction patterns
  • used to infer protein shape
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17
Q

Insulator

A
  • forms barriers between genes
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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
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19
Q

PCNA

A
  • homotrimeric

- prevents complex from disassociating from the strand, wrapped around strand

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20
Q

EF hand motif

A
  • binds calcium ions
  • acts like a hinge
  • helix-loop-helix pattern
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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
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22
Q

Restriction Endonucleases

A
  • cuts dsDNA at a specific palindromic sequence, in a symmetrical manner, sticky or blunt
  • methylated DNA is protected
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23
Q

UPR

A
  • Unfolded protein response
  • leads to the production of more chaperone proteins
  • in extreme case, leads to cell death
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24
Q

Epitope

A

Location on antigen which antibody binds to

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25
Q

Eukaryote Transcript Parts

A
  • 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
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26
Q

CRM

A
  • 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
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27
Q

Bacteria Expression Vectors

A
  • 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)
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28
Q

cDNA Libraries Purpose

A
  • goal is to represent mRNA in a given sample
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29
Q

Taq

A
  • DNA Polymerase
  • very from organism that lives in hot springs
  • thermostable
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30
Q

Drosophila Hsp90 Experiment

A
  • Hsp90 was knocked out by half in flies

- genetic variants became much more apparent, including cancers, discolourations

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31
Q

Phosphorylation

A
  • transfer of a phosphate group to a phosphoacceptor from the gamma phosphate of a NTP
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32
Q

Zinc Finger

A
  • motif that binds a zinc ion using histidine and cysteine residues
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33
Q

Protein Phosphatase

A
  • removes phosphate from a particular substrate
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34
Q

Transient Transfection

A
  • insert plasmid that contain viral ORI into cells
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35
Q

PCR Ingredients

A
  • 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
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36
Q

LC-MS-MS

A
  • 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
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37
Q

Dissassociation Constant

A
  • Kd

- ratio of [A][B]/[AB], if AB is a protein complex of A and B

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38
Q

Amyloid plaques

A
  • aggregates of misfolded proteins, causing cell death

- associated with Alzheimer Disease, Parkinson’s, ALS

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39
Q

Hsp ATP cycle

A

ATP bound - open, exposing hydrophobic domain
ADP bound - closed, protein allowed to fold
- multiple cycles are sometimes needed to fully fold protein

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40
Q

Dideoxy Chain-Termination Method and Ingredients

A
  • 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)
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41
Q

PCR Process

A

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

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42
Q

Phosphorylation motif

A
  • motif recognized by kinase

- contains free OH group, the phosphoacceptor

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43
Q

Ubiquitination

A
  • 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
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44
Q

Replication fork

A
  • aka growing fork

- formed at point where DNA duplex is currently being unwound

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45
Q

ddNTP

A
  • dideoxyribonucleoside triphosphate

- chain terminator, cannot be used to elongate chain further

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46
Q

Initialization Complex for RNA Transcription

A

RNA polymerase
General transcription factors
Mediator complex

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47
Q

Cyclin Dependent Protein Kinases

A
  • needed to cycle through cell stages
  • uses cyclin to confer substrate specificity
  • cyclins are polyubiquinated to make way for next ones
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48
Q

Gene

A

DNA necessary for synthesis of a functional product (polypeptide or RNA)
- usually entire transcription unit (including coding and regulatory DNA)

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49
Q

GTP Binding Proteins

A
  • 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
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50
Q

Gap repair

A
  • Ribonuclease H and FEN 1 remove RNA
  • Pol delta replaces RNA with DNA
  • DNA Ligase ligates them
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51
Q

Western Blot

A

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
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52
Q

Polyclonal Antibodies

A
  • 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
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53
Q

Immunostaining Proteins in situ

A
  • 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
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54
Q

Promoter

A
  • region of DNA upstream of gene acting as on-off switch

- not translated

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55
Q

Leading strand synthesis complex

A

Pol epsilon
RFC
PCNA

56
Q

Ion Exchange Chromatography

A
  • differentiate proteins based on charge (pI)
  • used charged beads
  • elute with competing ions, ex NaCl
57
Q

Genome

A
  • 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
Q

GFP

A
  • Green Fluorescent Protein
  • beta-barrel structure, with an oxygen binding domain inside the barrel
  • shines green when flash blue light
59
Q

Gene Families

A
  • 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
Q

Adenine Forms

A

Nucleoside - Adenosine
Nucleotide - Adenylate
dATP - deoxyribose adenosine triphosphate
rATP ribose adenosine triphosphate

61
Q

RNA Structures

A 
Hairpin: 
  - small loop, secondary
Stemloop: 
  - large loop, secondary
Pseudoknot: 
  - 2 loops emanating from a 2 stems, tertiary
  - part of function of telomerase
62
Q

Secretory Pathway

A
  • nascent polypeptides are earmarked for ER

- ribosome is bound to ER, and protein is translated directly into ER lumen, and modified concurrently

63
Q

Pol alpha

A

DNA Polymerase alpha

- elongates primer on lagging strand

64
Q

Immunoprecipitation (IP)

A
  • analytical analog to affinity chromatography

- uses affinity beads to a target protein, which sediment first when centrifuged

65
Q

DNA Barcoding Sequence Candidate

A
  • must lack introns, insertions, deletions, in order to easily be aligned with reference genomes
  • one candidate is CO1 inside mitochondria genome (mtDNA)
66
Q

DNA Helicase

A
  • 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
Q

Hsp70

A
  • monomer molecular chaperone

- most characterized Hsp molecule

68
Q

Gene Cluster

A
  • dense region of DNA containing genes of the same family
69
Q

Beta Sheet

A
  • interact laterally. Either parallel or antiparallel.

- can be used to form beta-barrels and beta-propellers

70
Q

RFC

A
  • Replication Factor C

- loads the DNA polymerase complex onto the template

71
Q

Lagging strand

A
  • unwound from 3’ to 5’, and synthesized from 5’ to 3’, so synthesis needs to happen discontinuously
72
Q

Amino Acid

A
  • has N-terminal and C-terminal
  • forms polypeptides
  • L (levo) stereoismer dominant in life
73
Q

Different Strands wrt. Transcription

A

Sense (coding strand)

Anti-Sense (template strand)

74
Q

DNA Forms

A

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
Q

Transfection

A

Introduce a gene into a eukaryotic cell

76
Q

SSR Types

A
  • 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
Q

Complex Transcription Unit

A
  • multiple possible gene products using alternative splicing

- a majority of genes in eukaryotic organisms

78
Q

UTR

A

untranslated region (of DNA)

79
Q

Trypsination

A
  • apply Trypsin protease, which cuts polypeptide into many chains
  • preparatory step to LC-MS-MS
80
Q

Next Generation Sequence

A
  • 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
Q

Temperature of DNA Melting

A
  • 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
Q

trans-regulatory module

A

regulates genes by producing proteins that in turn bind to the CRM

83
Q

Pseudogenes

A
  • inactive genes, remnants of once-duplicated genes
84
Q

Co-Immunoprecipitation

A
  • same as immunoprecipitation, but done more gently, in order to also analyze proteins that form complexes with target
85
Q

Gel Filtration Chromatography

A
  • 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
Q

Hsp90

A
  • dimer molecular chaperone
  • has ATP cycle
  • more specific to signalling molecules than Hsp70
87
Q

Heat Shock Proteins

A
  • aka HSP

- associated with cellular stress response

88
Q

Affinity Chromatography

A
  • use affinity beads in a chromatography column

- elute with acidic buffer to make beads release protein

89
Q

Ephrins

A
  • 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
Q

Supercoils

A
  • coils of coils
  • causes torsional stress
  • forms on parental DNA duplex ahead of replication coil
  • resolved with topoisomerases
91
Q

ampr

A
  • gene conferring ampicilin resistance

- often used in engineered plasmids for selection

92
Q

Origin of Replication

A
  • target for ORC
  • usually rich in AT
  • not very conserved
  • eukaryote chromosomes have many
93
Q

ORC

A
  • Origin Recognition Complex
  • found in eukaryotes
  • binds to origins of replication and associates with other proteins to load helicase
  • six subunit protein
94
Q

Protein Kinase

A
  • phosphorylates a particular substrate

- binds to a particular nucleotide (ex: ATP, GTP)

95
Q

RPA

A
  • 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
Q

Plasmid

A
  • circular extrachromosonal dsDNA, typical of bacteria & lower eukaryotes
  • replication occurs before cell division, decoupled from DNA genome replication
97
Q

SDS-PAGE

A
  • 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
Q

Prion Proteins

A
  • 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
Q

Protein Tertiary Structures

A
  • 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
Q

BiP

A
  • 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
Q

Popular Restriction Enzyme

A

EcoR1, sticky ends, from E-coli

102
Q

IEF

A
  • isoelectric focusing
  • apply charge to gel with pH gradient
  • molecules accumulate where charge is similar
103
Q

Homolog Types

A

Ortholog - across species

Paralog - in same species

104
Q

Large-T Antigen

A
  • hexomer

- a highly efficient helicase protein, powered by ATP, found in SV40 genome

105
Q

Disulfide Bridges

A
  • cysteins are bound together in ER by PDI, Protein Disulfide Isomerase
  • PDI is oxidized, or “reset” by Ero1
106
Q

Dideoxy Chain-Termination Method Process

A
  • denature and separate daughter strands by electrophoresis (with original DNA template eliminated)
  • uses polyacrylamide gel as opposed to agarose
    • more precise
107
Q

26S Proteasome

A
  • 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
Q

Motif

A
  • chain of polypeptides usually no longer than 40 residues

- confers a specific function to protein

109
Q

Rate Zonal Centrifugation

A
  • spin protein mixture in density gradient of sucrose (or other)
  • proteins of similar mass (S units) collect in bands
110
Q

General DNA Shape

A
  • 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
Q

Differential Centrifugation

A
  • run lysate sequentially in centrifuge
  • in between spins, collect supernatant and pellet
  • faster spinning puts lighter and lighter components in the pellet
112
Q

Coiled coil

A
  • motif containing alternating hydrophobic/hydrophilic residues
  • example: leucine zipper
  • polypeptide helices coiled around eachother
113
Q

cDNA Library Creation Process

A
  • 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
Q

Electropherogram

A

Used to validate base calling of Dideoxy Chain-Termination DNA sequencing

  • some regions are hard to read (repeats)
  • contamination might show overlapping peaks
115
Q

Lagging strand synthesis complex

A
  • 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
Q

ORF

A

Open Reading Frame

- coding region of DNA

117
Q

PCR Goal

A
  • polymerase chain reaction

- amplifies a DNA fragment

118
Q

Stable Transfection

A
  • done when there’s a protocol available to fuse gene of interest into target cell genome
119
Q

DNA Barcoding

A
  • used to identity species uniquely, accurately and quickly

- sensitive enough to differentate sequence between species, but not between mutations in the same species

120
Q

Translational Fusion Proteins

A
  • 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
Q

Michaelis Constant

A
  • Km
  • concentration of substrate when rate of reaction is half of Vmax
  • always same for same enzyme-substrate relation
122
Q

Pol epsilon

A

DNA Polymerase epsilon

- synthesizes leading strand

123
Q

Svedburg Units

A
  • location of molecule in density gradient after spun in a centrifuge
  • proportional to mas
124
Q

Polylinker

A
  • DNA region containing sites that are recognized by restriction endonucleases/enzymes
  • can insert foreign DNA into it to form recombinant plasmid
125
Q

SSR

A
  • Simple Sequence Repeats
  • sometimes used in DNA fingerprinting by looking at # of repeats (by PCR/electrophoresis)
  • expansion can be caused by backwards slippage
126
Q

Transcription Unit

A
  • exons, control regions, and Poly(A)/G-cap of a particular gene
  • control regions typically within 50kb upstream
127
Q

NGS Sequencing Pipeline

A
  • 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
Q

Leucine Zipper

A
  • 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
Q

Calmodulin

A
  • multifunctional calcium modulated protein

- binds calcium on an EF hand motif

130
Q

DNA Libraries

A

mixture of all the DNA fragments from an entire genome inserted into recombinant plasmids

131
Q

Polyubiquitination

A
  • tag a protein for death
  • ubiquitin proteins attached a Lysine residue on protein, in a chain
  • monoubiquitin does not tag cell for death
132
Q

Affinity Bead

A
  • Agarose Bead -> Protein A -> Fc domain of IgG antibody

- antibody used depends on which protein is targeted

133
Q

Enzyme Structure and Catalysis

A
  • binding site/pocket
  • catalytic site/cleft
  • lowers energy of transition state required for reaction to occur
134
Q

Chaperonin

A
  • type of chaperone
  • very large protein
  • highly conserved
    • TriC in humans
    • GroEL, GroES in bacteria
  • has ATP cycles
135
Q

Hemoglobin

A
  • tetramer made up of 2 alpha units, 2 beta units

- beta unit is highly conserved

BIOL 200 (3 decks)

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