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Final Review - Bureau Lectures

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Biology (Sci)
BIOL 200
Mathieu Roy

PROKARYOTIC TRANSCRIPTION (DNA -> MRNA)  DNA template o serve as a blueprint for the RN sequence  ribonucleotides  serve as monomers for RNA polymerixation rna polymerase o DNA dependant RNA polymerase catalyze the synthesis of RNA INITIATION 1. RNA polymerase binds to the promoter region a. AT rich => facilitates the melting of the two strands 2. polymerase melts duplex dna near the transcription start site a. transcription bubble/open complex 3. polymerase catalyzes the phosphodiester linkage of two initial rNTP's TRNASCRIPTION  prokayrotes o compact, circular genomes o genes of similar function are grouped together o no nucleus (genome sits in the periplasm)  transcribe the entire RNA o trp mRNA: start sites for protein synthesis (POLYCISTRONIC mRNA)  rarely happens in eukaryotes  no modular format  all on the same promoter o eukaryotes: different promoters  operons - EUKARYOTIC TRANSCRIPTION  results in multiple mRNA's: specific for each protein  theoretically one ribosome to transcribe each mRNA POST-TRANSCRIPTIONAL MODIFICATION  addition of the 5' cap  addition of the 3' poly-A tail  splicing DNA REPLICATION  semi-conservatice  performed by DNA polymerase o DNA dependent DNA polymerase  requires dNTP's  requires a primer (DNA or RNA)  proceeds in a 5' -> 3' direction PROKARYOTIC REPLICATION  TOPOISOMERASE: relieves supercoils o found in prokaryotes & eukaryotes o binds upstream to the replication fork (far enough away to do it's job)  HELICASE: unwinds duplex DNA o rate limiting step in DNA replication  LEADING STRAND SYNTHESIS o primer catalyzed by o DNA dependent DNA polymerase  LAGGING STRAND SYNTHESIS o -dna Is elongated from an RNA primer (formed by primase) by DNA polymerase o OKAZAKI FRAGMENTS: hybrid of DNA & RNA  get rid of RNA component with RNAsH  two adjoining molecules are ligated together by DNA ligase ENDONUCLEASE: anything that cuts in the middle EUKARYOTIC DNA POLYMERIZATION SV40 as a model of eukaryotic DNA replication  LARGE T-ANTIGEN o 6 subunits which are identical to eachother o helicase: very fast  able to replicate DNA much faster o encoded by the viral genome  as the large t-antigen  REPLICATION PROTEIN A (RPA) o binds single stranded DNA o keeps DNA in optimal conformation for copying by DNA polymerase o PROTEIN: single stranded binding protein (SSbP)  DNA POLYMERASE DELTA (POL DELTA) o proofreading activity  replication factor C  proliferating cell nuclear antigen (PCNA) o carry out leading strand DNA syntehsis o RPA is displaced as sysnthesis proceeds  primase/pol-alpha complex o primase formats the RNA componenet of the primer and DNA polymerase.... o on leading + lagging strand  pol delta/ rfc / PCNA complex prokaryotes - only have primase eukaryotes - have primase + pol delta DNA POLYMERASE ALPHA - make a dna primer for the okazaki fragment o only occurs in eukaryotes RIBNUCLEASE H & FEN-1  displace the RNA componenet at the 5' ends of the okazaki fragments after ribonuclease H & FEN 1  polymerase delta replaces RNA with DNA  DNA fragments are then ligated together by DNA ligase EXONUCLEASES AND ENDONUCLEASE EXONUCLEASES - liberate single nucleotides at either end ENDONUCLEASES - cleaves phosphodiester bonds with a polynucleotide … o sticky or blunt ends SOME DNA POLYMERASES HAVE A PROOFREADING ACTIVY  motifs which have different functions  polymerase: adds n.t. to the growing DNA polymer  polymerase delta (not alpha) o exonuclease (3' -> 5' proofreading activity) BASE EXCISION REPAIR (BER)  5-methyl cytosine can undergo spontaneous deamination to thymine o causes unmatched base pairs 1. DNA GLYCOSYLASE  hydrolyze the bond between the mispaired base and the sugar-phosphate backbone 2. APE1 cuts the dna back bone o endonuclease 3. AP LYASE associated with DNA polymerase Beta remoces the deoxyribose __ 4. dna polymerase beta fills the gap and dna ligase seals the nick in the sugar-phosphate backbone MISMATCH EXCISION REPAIR  error incorporated into the newly synthesized strand  MSH2 & MSH6 bind to the daughter strand  triggers bind of the activity of MLH1 ENDONUCLEASE o recruits PMS2 o endonuclease has to make the initial cut, so exonuclease can come and function  DNA HELICASE unwinds and DNA EXONUCLEASE digests the segment of the duaghter strand  gap repair by POL DELTA and DNA LIGASE DS BREAK REPAIR STRAND BREAK REPAIR BY HOMOLOGOUS RECOMBINATION DNA CLONING  take advantage of endonucleases  EcoR1 - endonuclease which makes sticky end cuts recombinant plasmid put into baceteria bacteria take up the plasmid and replicate the plasmid  the cateria POLYMERASE CHAIN REACTION (PCR) reaction tube contents  DNA template  oligonucleotide primes  Taq DNA polymerase o can use other DNA dependent DNA polymerase  dNTP's thermal cycler: programmable heat block CYCLE 1 - repeat cycle 1 (20-40 times) a. DENA
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