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Biochemistry II, DNA, RNA

by: ChasePrater

Biochemistry II, DNA, RNA 4510

Marketplace > Middle Tennessee State University > 4510 > Biochemistry II DNA RNA
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About this Document

These are notes taken on April 12 and 14th.
Biochemistry II
Dr. Ooi
Class Notes
biochemistry, DNA, RNA, nucleotide, mutation
25 ?




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This 4 page Class Notes was uploaded by ChasePrater on Friday April 15, 2016. The Class Notes belongs to 4510 at Middle Tennessee State University taught by Dr. Ooi in Spring 2016. Since its upload, it has received 11 views.


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Date Created: 04/15/16
April  12  &14     th   DNA  Packaging:     -­‐ DNA  is  very  large  (human  genome  is  about  3  billion  base  pairs),  but  has   to  packaged  in  a  very  small  cell.     -­‐ Chromatin  is  packaged  to  form  chromosomes.  Nucleosomes  get  packaged   into  a  small  fiber,  then  that  is  coiled  further  into  filaments.   DNA  Properties:   -­‐ Hyperchromic  effect:  denatured,  single  stranded  DNA  absorbs  light  much   better  than  native  double  stranded  DNA.   -­‐ Melting  Curve:  at  half  of  the  maximum  absorbance  on  an  absorbance  vs   temperature  graph,  this  midpoint  is  the  “melting  temperature”.  This   depends  on  ion  concentration  (  salt  ins  stabilize  negative  charges  on  DNA   and  increase  melting  point),  pH  of  solution,  and  mole  fraction  of  G-­‐C  bp.     -­‐ Denatured  DNA  can  renature  if  it  is  cooled  back  below  its  melting  point.     Nucleic  Acids  &  stabilization   -­‐ Stacking  interactions:  they  are  hydrophobic  &  van  der  Waals  forces   between  aromatic  portions  of  adjacent  bases.  G-­‐C  interactions  are  greater   than  A-­‐T.   -­‐ Cations  help  to  stabilize  the  negative  charges  on  DNA  backbone,  which   reduces  the  amount  of  repulsion  and  allows  DNA  to  be  coiled  tighter.   Mg2+  is  essential  in  stabilizing  RNA.   RNA   Forms  and  functions:     -­‐ mRNA  and  viral  RNA  for  info  storage.   -­‐ tRNA  for  info  transfer.   -­‐ Ribozymes  for  catalytic  properties.   -­‐ rRNA  for  Ribosomes   Structure:   -­‐ single  stranded  but  can  fold  into  complex  structures.   -­‐ Can  fold  over  to  form  hairpin  structure.   -­‐ Can  form  helices  with  itself.     mRNA   -­‐ carry  info  for  protein  synthesis   -­‐ made  during  transcription,  and  secondary  structure  is  unique  to  each   mRNA.     -­‐ Polycistronic  in  prokaryotes  meaning  that  one  strand  carries  many  genes.   This  happens  because  their  DNA  does  not  introns  between  the  genes.     -­‐ Monocistronic  in  eukaryotes  meaning  that  each  mRNA  carries  only  one   gene.   -­‐ Contain  a  poly  A  tail,  which  keeps  it  from  being  broken  down   immediately,  and  it  also  helps  it  leave  the  nucleus.     tRNA   -­‐ small,  about  70-­‐100  nucteotides.   -­‐ Contains  hairpins  creating  4  loops  and  4  helical  stems.   -­‐ Acceptor  stem  bind  to  amino  acid.   -­‐ D-­‐loop  contains  a  dihydrouracil.   -­‐ Anticodon  loop   -­‐ TGammaCG  loop  contains  an  invariant  sequence,  and  a  pseudouracil  that   helps  the  ribosome  recognize  it.   -­‐ Variable  loop  that  is  different  depending  on  the  organism..   -­‐ Stabilized  by  H  bonds  between  D  and  TGamaCG.   -­‐ Contains  nonstandard  H-­‐bonds  such  as  G-­‐U,  and  3  base  bonding.     rRNA   -­‐ 65%  or  ribosomes  are  RNA.   -­‐ Highly  folded  pattern,  lops  with  U-­‐turn  motive,  and  internal  loops.     -­‐ Secondary  structure  is  conserved  between  all  kingdoms,  so  it  is  used  to   compare  and  classify  organisms  and  make  the  phylogenetic  tree.  It  is   useful  because  all  organisms  have  them,  they  are  large  enough  to  look  at,   and  are  highly  conserved.     Ribozymes   -­‐ RNA  molecules  that  act  as  enzymes.     -­‐ They  are  considered  “intruders”  in  protein  kingdom  even  though  they   came  before  proteins.   -­‐ Contain  a  catalytic  motif  for  the  site  of  cleavage  and  a  flanking  sequence   to  help  guide  ribosome  to  mRNA.     -­‐ Hammerhead  ribozyme  can  cleave  itself.     -­‐ May  be  used  in  medicine  to  cleave  mRNA  that  is  carrying  genes  that  are   for  building  bad  proteins.     DNA  vs  RNA     -­‐ RNA  came  first,  but  DNA  was  developed  as  a  safer  way  to  store  genetic   info.       -­‐ The  oldest  RNA  is  reverse  transcriptase.     Non-­‐Watson-­‐Crick  base  pairing   -­‐ Hoogsteen  pairing:  A  &  T  form  h  bond,  but  on  the  wrong  side  of  the  A.   -­‐ U-­‐C  pair,  and  A-­‐A  pairing.  These  are  not  mutations.     Isolation  of  Nucleotides   Agarose  gel  Electrophoresis   -­‐ restriction  enzymes  chop  DNA  into  fragments.   -­‐ The  fragments  are  separated  based  on  size.  The  bigger  fragments  have  a   harder  time  moving  through  the  gel  as  the  electrical  current  tries  to  pull   them  to  the  bottom.     -­‐ At  the  end  of  the  run  the  gel  is  stained  with  ethidium  bromide  and   observed  under  UV  light.     -­‐ Ethidium  bromide  is  a  very  dangerous  stain  because  it  permanently   lodges  itself  in  the  helix  of  DNA.     -­‐ Intercalaing  agents:  ethidium,  proflavin,  and  acridine  orange.   Nucleic  Acids  Technique   -­‐ Hybridizaton  is  a  technique  used  to  ID    a  specific  gene  by  Southern   blotting.     -­‐ Bands  of  DNA  from  gel  are  transferred  to  nitrocellulose  paper.  DNA  must   first  be  soaked  in  0.5M  NaOH  to  make  it  single  stranded.     -­‐ A  probe  is  attached  to  a  small  complimentary  fragment  that  will  bind  to   the  gene  of  interest.   -­‐ After  membrane  and  probe  are  incubated  together  they  are  washed  off.   -­‐ Because  the  probe  is  radioactive  it  can  be  seen  under  x-­‐ray.     -­‐ Northern  blotting  is  for  RNA.   -­‐ Western  blotting/immunoblotting  is  for  proteins.   Mutations   Spontaneous  mutations   -­‐ transition  mutations  from  incorrect  base  pairing  due  to  tautomerizartion   of  purines.   -­‐ Hydrolytic  reactions:  Depurination  when  a  glycolase  chops  off  a  base   from  the  sugar  leaving  an  APsite/  ABasic  site.  Deamination  can  turn  C   into  U.     Mutations  due  to  external  factors   -­‐ Ionizing  radiation:  x-­‐rays  and  gamma  rays  can  break  strands  and  cause   cross  linking.  UV  light  can  only  cause  cross  linking.   -­‐ Xenobiotics:  analogs  that  look  like  nucleosides  and  are  incorporated  into   DNA.   -­‐ Alkylating  agents:  adding  alkyl  groups  to  A  and  G.     -­‐ Non-­‐alkylating  agents:  deamination,  possibly  caused  by  nitrous  acid.   -­‐ Intercalating  agents:  planar  molecules  that  get  stuck  between  DNA   strands.     Point  mutation   -­‐ any  type  of  single  base  alteration.   -­‐ Deletion  or  insertion  of  a  single  bas  is  the  worst  because  it  may  mess  up   reading  frame.     -­‐ Substitution     -­‐   Transition  mutation:  swap  a  purine  for  a  purine  and  pyr  for  pyr.   -­‐   Transversion  mutation:  swap  a  purine  for  pyrimidine  and  vice  versa.   -­‐ Silent  mutation:  one  base  is  switched    out  resulting  in  no  change  in  the  AA   sequence.  Generally  switching  the  3  position  of  an  amino  acid  codon   does  not  change  the  AA  that  it  codes  for.     -­‐ Lethal  mutation:  a  single  base  mutation  that  causes  an  essential  AA  to  be   replaced  by  another  AA,  which  creates  inactive  protein.     Repair  mechanisms   -­‐ Excision  repairs:  damaged  section  of  DNA  is  removed  and  replaced.  2   types.   -­‐   1.  Nuclease  excision:  nuclease  cuts  out  bad  bases  and  polymerase  I     fills  in  gaps  and  ligase  joins  segments.   -­‐   2.  Bad  base  is  removed  by  glycosylase  resulting  in  an  APsite.  AP     endonuclease  removes  the  empty  sugar  and  polymerase  I  fills  gaps     and  ligase  joins  pieces.     -­‐ Photo-­‐reactivation  repair:  photoreacting  enzyme  is  activated  by  visible   light  and  cleaves  C-­‐C  cross-­‐linkage  bond.     -­‐ Recombinational  Repair:  last  minute  rescue.  Stops  replication  and  starts   it  again  after  the  bad  bases,  which  creates  a  gap.  Gap  is  filled  by   exchanging  pieces  of  homologous  DNA.          


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