MCB 250 EXAM I STUDYGUIDE
MCB 250 EXAM I STUDYGUIDE MCB 250
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This 13 page Study Guide was uploaded by Jessica Logner on Sunday May 15, 2016. The Study Guide belongs to MCB 250 at University of Illinois at Urbana-Champaign taught by Kirchner, N in Spring 2016. Since its upload, it has received 7 views.
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Date Created: 05/15/16
MCB 250 EXAM I Electronegativity Trend Upper right diagonal corner Ionic Bonds opposite charges Hydrogen Bonding weak bond (dipoledipole) with nitrogen or oxygens Van der Waals force between two molecules not covalently bonded over a large surface Hydrophobic nonpolar molecules Lipid head hydrophillic fatty acid tails hydrophobic Single bonds can rotate Double bonds, Triple, or Resonance rigid stereoisomers can't superimpose mirror with actual image acids H donors () Bases H acceptors (+) For acids 0 if pKa is above pH and if pKa is below pH For bases 0 if pKa is below pH and + if pKa is above pH deprotonating more basic aka decreasing H+ 5' end phosphate group 3' end OH group What is responsible for DNA structure? base pairing base stacking hydrophobic interactions of bases repulsion of negatively charged phosphate groups B DNA structure 20 A diameter 34 A per helical turn 10 bp per helical turn bases rotate every 36 degrees Major groove 22 A contains more chemical info if on plane of paper, hump is above plane (bases above backbone) Minor groove 12 A if on plane of paper, hump is below plane (bases below backbone) Denatures High temp Urea increases (blocks H bonds) Methanol increases (disrupts phobic interactions) pH increases decrease in salt concentration Renaturation slow cooling aka time increases increasing salt concentration concentration of DNA increases base pair complexity Palindrome same sequence as its complementary strand write complementary and read both 5' to 3' 5' overhang cut is close to 5' end and the complementary strand has long 3' to 5' below template agarose broad separation range acrylamide narrow separation range PCR 1 cycle = 2 copies dsDNA 2 cycles = 4 copies dsDNA 3 cycles = 8 copies dsDNA ` peptide bond contains carbon double bonded to oxygen and single bond with NH (rigid) primary structure linear with N terminus on left and C terminus on right secondary structure folded residues formed by hydrogen bonding of atoms among the polypeptide backbones (alpha and beta sheets) tertiary structure involves interaction of r group side chains weak noncovalent interactions covalent disulfide bonds quarternary structure interaction of multiple polypeptide chains and protein subunits Protein folding can take place as the protein is being synthesized N terminal folds before C terminal Protein Folding Chaperones groE (ATP dependent) peptidylproline isomerases (transforms trans to cis) disulfide bond isomerases (finds most energetically favored state) PAGE denatures protein with SDS and reducing agent depends on mass IEF pI = net charge of pH is 0 depends on pI 2DPAGE both IEF and SDS (mass and pI) Column Chromatography larger proteins move quicker proteins are eluted by altering salt concentration Writhe negative if RH turn Unwinding DNA increases amount of bp per turn If lk changes covalent bonds are broken Type II Topos ATP dependent passes both strands though another dsDNA Changes LK to +2 (Topo IV) DNA gyrase changes LK to 2 (Topo II) does this by adding negative supercoils (aka changing wr number) Type I Topos relaxes DNA by decreasing supercoils ATP independent (uses phosphate backbone) passes one strand through the other changes LK to +1 does this by adding a helical turn Histone protein very basic (lysine and arginine) therefore binds well with negative backbone of DNA Beads on String 11nm H1 binds linker DNA forming fibers 30 nm (stabilized by N terminal tails that stick out) Histone tails can be methylated, acetylated, or phosphorylated these modifications can affect function and decide whether a gene gets expressed or not synthesizing DNA 5' to 3' (direction of leading strand) leading strand template is 3' to 5' lagging strand template is like forming leading strand so 5' to 3' lagging strands are formed 5' to 3' which is why they become okazaki fragments fork moves to the right when 3' is on the top left on ds DNA Helicase donut melts parental dsDNA interacts with Pol III and Primase hexamer with 6 subunits wraps around lagging strand template and travels 5' to 3' recruits primase and Pol III by interacting with tau proteins helicase loader uses ATP to load the helicase SSB binds ssDNA template prevents reannealing Primase synthesizes RNA primers recruited by helicase starts at 5'CTG3' and puts down 1012 RNA bases DNA Pol III core performs 3' to 5' exonuclease (corrects mistakes) has to recognize tau proteins (ter sites are bound by tus proteins) clamp loader recognizes 3' end of primer (end of primer) and uses ATP to open the clamp clamp recruits Pol III and has 35 A hole big enough for B DNA and doesn't bind to DNA (increases processivity) RNAse H removes RNA primer from hybrid leaves one ribonucleotide DNA Pol I polymerase activity 3' to 5' exonuclease (corrects mistakes) 5' to 3' exonuclease (remove RNA or DNA) starts at end of Pol III and fills gap with dNTPs DNA ligase seals nicks between okazaki fragments Dna A initiator protein of replication (multiple needed) IHF and FIS rich in Arg and Lys (positive amino acids) binds and bends DNA located on oriC What primes the leading strand? the lagging strands okazaki fragments telomerase used in eukaryotic chromosomal replication contains RNA that acts as template extends 3' end of chromosome to prevent it from continually shrinking as replication continues What prevents rapid reinitiating of DNA replication after it just replicated? methylation Dam recognizes sequence 5' GATC 3' methylates adenine 11 methylation sites in oriC Right after replication oriC is hemimethylated daughter cells only have 1/2 amount of initiation proteins (Dna A) What prevents rapid reinitiation of DNA replication after it just replicated? 1. more initiator protein (DnaA) has to be made 2. active form of the initiator (DnaAATP) has to be made 3. oriC site has to become fully methylated so the initiator (DnaAATP) can bind nucleoside vs nucleotide side doesn't have phosphate while tide has sugar, base, and phosphate ATP and GTP energy intermediates cAMP regulation and cell signaling NAD+ and FAD+ enzyme cofactors distance btwn bases 3.4 A SDS gets rid of secondary structure (in proteins not DNA) How does proline destabilize secondary protein structure? makes helicases stop curves back and attaches to amino terminal end base pair determination 10 x twist What interaction helps position Pol III near replication fork? A: helicase interacts with tau proteins What interaction helps position the primase? helicase positions primase DNA polymerase high fidelity exonuclease to increase accuracy DNA polymerase high processivity helicase number of nucleosomes length in kbp/ (linker + wrap around) micrococcal nuclease can only digest exposed DNA aka linker endonuclease restriction enzymes with recognition sites exonuclease recognizes backbone Helicase/Helicase loader 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 SSB binds single stranded DNA template prevents reannealing Primase synthesizes RNA primers Starts at 5' CTG 3' and lays down a 1012 RNA primer DNA Pol III 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 RNAse H Removes RNA primers Can only cleave bonds between ribonucleotides Therefore, leaves one ribonucleotide DNA Pol I 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 DNA ligase seals nicks between okazaki fragments Processivity Average number of nucleotides added each time enzyme interacts with DNA template Sliding clamp 35 angstrom completely surrounds DNA Does not bind to DNA Tethers DNA Pol III core to the DNA Confers processivity DnaA THE initiator protein ATPase Can bind and cleave ATP to ADP Multiple DnaA's are required Integration Host Factor (IHF)/Factor for Inversion Stimulation (FIS) Bends DNA for replication initiation Rich in Arg and Lys Histone like Ter Sites DNA sites with certain orientation where to replisome would stop 23 bp bound by Tus proteins Stops helicase activity Telomerase Special DNA polymerase Contains own RNA template Extends 3' end of chromosome Deoxyadenosine methylase (Dam) recognizes sequence 5' GATC 3' adds methyl group to adenine (11 sites in oriC) Acids proton donors Bases proton acceptors Nucleotide Sugar + phosphate + nitrogenous base Ribose Hydroxyl group on 2' carbon Deoxyribose H group on 2' carbon Pyrimidines Single ring Cytosine (DNA and RNA) Uracil (RNA) Thymine (DNA) Purines Double ring Adenine (DNA and RNA) Guanine (DNA and RNA) Nucleoside Sugar + base BDNA 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 Denaturation of DNA High temperature (High GC has higher Tm because 3 H bonds instead of 2) Hydrogen bonding reagents (urea) Methanol PH > 11 Decreased salt concentration Primary structure linear sequence of amino acids in protein Secondary structure folding of chain involving residues close together Formed by H bonding of atoms among backbone Alpha helix Beta strand Beta pleated sheets Tertiary structure Overall folding of the whole polypeptide Involves interaction of R group side chains (weak non covalent interactions and covalent disulfide bonds) Quaternary Structure Interaction of multiple polypeptide chains or protein subunits Peptidylproline Isomerase In presence of this isomerase, peptide bonds can switch between cis/trans Denaturation of a protein Temperature Hydrogen bonding reagents (urea) pH Methanol Detergents (SDS) Reducing reagents (DTT and mercaptoethanol) break disulfide bonds SDSPAGE 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 Isoelectric point (pI) the pH at which a protein has a net charge of zero Isoelectric Focusing (IEF) Electrophoresis in a pH gradient causes each protein to migrate to a point where pH=pI and stop separates based on pI 2D PAGE IEF and SDSPAGE combined Potential charged groups in proteins N and C terminal Side chains of Asp, Glu, Cys, Tyr, His, Lys, Arg Size Exclusion Chromatography 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 Ion Exchange Chromatography Column matrix has positive or negative charge How tightly protein sticks depends on total net charge Separating based on total net charge Linking Number (Lk) Twist (Tw) + Writhe (Wr) Total number of times DNA strands cross one another Twist (Tw) number of helical turns Writhe (Wr) Number of times helix crosses itself Type II Topoisomerase 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 doublestranded DNA Covalent intermediate 5' phosphate to tyrosine Changes linking number by 2 Type I Topoisomerase Changes twist Top I and Top III (odd numbers) Relaxes DNA, decreases number of supercoils Does not require ATP Makes singlestranded breaks and passes ONE strand through another Covalent intermediate 5' phosphate to tyrosine Changes linking number by +1 Chromatin DNA + proteins Histone Core 8 member disk 2 types of each H2A, H2B, H3, and H4 Nucleosome Core histones + DNA wrapped around 147 bp around histone core + 2060 linker DNA dsDNA wraps around core ~1.65 times H1 histones bind linker DNA to create 30nm fibers Histone tails protrude from nucleosome can be acetylated, methylated, or phophorylated
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