46 Cards in this Set
| Front | Back |
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Helicase/Helicase loader
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-Breaks hydrogen bonds of parental dsDNA
-interacts with Pol III and primase
-Helicase wraps around lagging strand template
-Travels 5' to 3'
-Uses ATP to unwind helix
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SSB
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-binds single stranded DNA template
-prevents reannealing
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Primase
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-synthesizes RNA primers
-Starts at 5' CTG 3' and lays down a 10-12 RNA primer
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DNA Pol III
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-DNA Dependent, DNA synthesizing
-synthesizes DNA starting at RNA primers
-Can only add bases to free 3' OH group so requires a primer
-3' to 5' exonuclease to correct mistakes
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RNAse H
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-Removes RNA primers
-Can only cleave bonds between ribonucleotides
-Therefore, leaves one ribonucleotide
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DNA Pol I
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-Removes RNA primers and fills gaps with DNA
-polymerase activity
-3' to 5' exonuclease activity to fix errors
-5' to 3' exonuclease to remove RNA or DNA
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DNA ligase
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seals nicks between okazaki fragments
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Processivity
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Average number of nucleotides added each time enzyme interacts with DNA template
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Sliding clamp
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-35 angstrom completely surrounds DNA
-Does not bind to DNA
-Tethers DNA Pol III core to the DNA
-Confers processivity
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DnaA
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-THE initiator protein
-ATPase
-Can bind and cleave ATP to ADP
-Multiple DnaA's are required
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Integration Host Factor (IHF)/Factor for Inversion Stimulation (FIS)
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-Bends DNA for replication initiation
-Rich in Arg and Lys
-Histone like
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Ter Sites
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-DNA sites with certain orientation where to replisome would stop
-23 bp
-bound by Tus proteins
-Stops helicase activity
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Telomerase
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-Special DNA polymerase
-Contains own RNA template
-Extends 3' end of chromosome
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Deoxyadenosine methylase (Dam)
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-recognizes sequence 5' GATC 3'
-adds methyl group to adenine (11 sites in oriC)
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Acids
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proton donors
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Bases
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proton acceptors
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Nucleotide
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Sugar + phosphate + nitrogenous base
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Ribose
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Hydroxyl group on 2' carbon
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Deoxyribose
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H group on 2' carbon
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Pyrimidines
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-Single ring
-Cytosine (DNA and RNA)
-Uracil (RNA)
-Thymine (DNA)
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Purines
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-Double ring
-Adenine (DNA and RNA)
-Guanine (DNA and RNA)
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Nucleoside
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Sugar + base
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B-DNA
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-Right handed
-Hydrogen bonded bases lie on same plane
-plane perpendicular to helix axis
-20 Angstroms in diameter
-34 Angstroms per helical turn
-10 or 10.5 bp per helical turn
-bases rotated 36 degrees in respect to each other
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Denaturation of DNA
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-High temperature (High GC has higher Tm because 3 H bonds instead of 2)
-Hydrogen bonding reagents (urea)
-Methanol
-PH > 11
-Decreased salt concentration
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Primary structure
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linear sequence of amino acids in protein
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Secondary structure
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-folding of chain involving residues close together
-Formed by H bonding of atoms among backbone
-Alpha helix
-Beta strand
-Beta pleated sheets
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Tertiary structure
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-Overall folding of the whole polypeptide
-Involves interaction of R group side chains (weak non covalent interactions and covalent disulfide bonds)
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Quaternary Structure
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Interaction of multiple polypeptide chains or protein subunits
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Peptidylproline Isomerase
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Peptidylproline Isomerase
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Denaturation of a protein
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-Temperature
-Hydrogen bonding reagents (urea)
-pH
-Methanol
-Detergents (SDS)
-Reducing reagents (DTT and mercaptoethanol) break disulfide bonds
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SDS-PAGE
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-PAGE= Polyacrylamide Gel Electrophoresis
-For movement to be proportional to mass, the molecule must have the same shape and a constant charge to mass ratio. This is accomplished by dissolving protein in SDS
-Separates based on mass
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Isoelectric point (pI)
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the pH at which a protein has a net charge of zero
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Isoelectric Focusing (IEF)
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-Electrophoresis in a pH gradient causes each protein to migrate to a point where pH=pI and stop
-separates based on pI
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2D PAGE
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IEF and SDS-PAGE combined
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Potential charged groups in proteins
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-N and C terminal
-Side chains of Asp, Glu, Cys, Tyr, His, Lys, Arg
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Size Exclusion Chromatography
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-column matrix with polysaccharide gel beads
-small proteins enter beads and are slower
-large proteins are excluded from beads and pass through column first
-Separating based on size
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Ion Exchange Chromatography
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-Column matrix has positive or negative charge
-How tightly protein sticks depends on total net charge
-Separating based on total net charge
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Linking Number (Lk)
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-Twist (Tw) + Writhe (Wr)
-Total number of times DNA strands cross one another
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Twist (Tw)
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number of helical turns
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Writhe (Wr)
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Number of times helix crosses itself
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Type II Topoisomerase
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-Changes writhe
-Top II and Top IV (even numbers), changes Lk by +2
-DNA Gyrase: only one that can make negative supercoils, changes Lk by -2
-Requires ATP
-Passes BOTH strands through another double-stranded DNA
-Covalent intermediate 5' phosphate to tyrosine
-Changes linking numbe…
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Type I Topoisomerase
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-Changes twist
-Top I and Top III (odd numbers)
-Relaxes DNA, decreases number of supercoils
-Does not require ATP
-Makes single-stranded breaks and passes ONE strand through another
-Covalent intermediate 5' phosphate to tyrosine
-Changes linking number by +1
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Chromatin
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DNA + proteins
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Histone Core
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-8 member disk
-2 types of each
-H2A, H2B, H3, and H4
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Nucleosome
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-Core histones + DNA wrapped around
-147 bp around histone core + 20-60 linker DNA
-dsDNA wraps around core ~1.65 times
-H1 histones bind linker DNA to create 30nm fibers
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Histone tails
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-protrude from nucleosome
-can be acetylated, methylated, or phophorylated
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MCB 250: Exam 1