LECTURE 2 REPLICATION AND TRANSCRIPTION DNA by pptfiles

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									               LECTURE 2

REPLICATION AND TRANSCRIPTION
             DNA


           Endang S. Rahayu
       Fakultas Teknologi Pertanian
        Universitas Gadjah Mada
DNA Replication
Circular DNA is replicated bidirectionally
Origin of Replication in Eukaryotic
               DNA
Rolling circle replication
          DNA polymerase
• DNA polymerase I catalyses the covalent
  attachment of new deoxyribonucleotide
  units by their α-phosphate groups to the
  free 3’-hidroxil end of preexisting DNA
  chain
• The direction of synthesis of the DNA
  strand is thus from the 5’ end toward the 3’
  end
 Preformed DNA is necessary for the action
           of DNA polymerase
• The action of DNA polymerase needs : primer
  and template
• One strand of the preformed DNA serves as the
  primer and the other strand serves as a template
• The product of DNA polymerase is a base paired
  duplex
DNA replication requires many enzymes and
               protein factors
Replication takes place in many sequential steps including:
• Recognition of the origin
• Unwinding of the parent duplex
• Holding the template strand apart
• Initiation of the new daughter strands
• Elongation of the daughter
• Rewinding
• Termination
Replisome – DNA replicase system
            DNA polymerase of E. Coli

                           I        II        III
Activity catalyzed
5’-3’ polymerization       +         +         +
5’-3’ exonuclease          +         -         +
3’-5’ exonuclease          +         +         +
Molecular weight        109.000   120.000   400.000
Molecular per cell       400                  10
Activity (nucleotides    600        30       9.000
per minute per
molecule at 37ºC
    Simultaneous replication of DNA

• Two strand of DNA replicate simultaneously at
  replication fork
• One end growing strand would have its 3’ end
  but the other strand would have its 5’ end
• At the 3’ end polimerase can add new nucleotide
  to the other end polimerase can not add the new
  nucleotide
           Okazaki fragment
• What is okazaki fragment?
• Leading strand – one strand of DNA is
  replicated continously in the 5’ – 3’
  direction
• Lagging strand – other strand is made
  discontinously in short pieces, also by
  adding new unit to 3’ end
Okazaki fragments
Synthesis of okazaki fragments
 The okazaki fragments are spliced
         by DNA ligase

• The new okazaki fragments is joined to the
  lagging strand by the enzyme DNA ligase :
  which form a phosphodiester bond
  between 3’ – OH group at the end of the
  DNA undergoing elongation and the 5’ – P
  group of the newly made okazaki fragment
Replication requires physical separation of
          the parent duplex DNA
Proofreading activity
Replication in eukaryotic cells
   Genes as transcribed to yield
              RNAs
• Transcription – when gene is transcribed
  to RNAs (mRNA, tRNA, rRNA)
• RNA strand having a base sequence
  complementary to one of the DNA
• During replication the entire chromosomes
  is copied to yield daughter DNA identical
  to the parent
• During transcription – a part of DNA is
  transcribed
 Messenger RNA code for polipeptide
             chains
• mRNA are long single strand molecules
• Monocistronic (monogenic) – mRNA that
  carries the code for only one polipeptide
• Polycitronic (polipegic) – carries two or
  more different polipeptide
  Eukaryotic cell nuclei have three
        RNA polymerases
• Table 2. Product of eukaryotic DNA - directed
  RNA polymerase

Enzyme               Product                Location
RNA polymerase I     5.8S, 18S, 28 rRNAs    Nucleolus
RNA polymerase II    mRNA                   Chromatin
                                            and
RNA polymerase III tRNAs, 5R rRNA
                                            nucleoplasm

• Mitochondria contain only a single RNA
  polymerase
 DNA – directed can be selectively inhibited

• Actinomycin D – by intercalation which
  producing a deformation in the DNA template
• Acridin – by intercalation
• Rifampicin – bind to the ß sub unit of RNA
  polimerase of prokaryotes and prevent the
  initiation of RNA synthesis
• α – amanitin inhibits RNA synthesis in
  eukaryotes by blocking the RNA polimerase II
          Ekspresi Gen Eukaryot

 Enhancer             Promoter Transcribed Region Terminator

                          Transkripsi             RNA Polymerase II
          Transkrip Primer 5’           Intron1           3’
                                Exon1             Exon2
                                                  Cap
                                                  Splice
Translasi                                         Cleave/Polyadenylate
                             7mG                   An
      N
  C                                           Transport
                              7mG                  An
Polipeptida
Ekspresi Gen Prokaryot

Promoter Cistron1           Cistron2 CistronN Terminator

          Transkripsi              RNA Polymerase
mRNA 5’                                             3’
                      1        2          N
                                   Ribosom, tRNAs,
          Translasi
                                   Protein Factors
                           N             N
              N                    C
          C
                                          C
                  1            2         3
                          Polipeptida
Heterogenous nuclear RNAs are precursors
      of eukaryotic messenger RNAs

• Eukaryotic mRNA are monogenic, many
  prokaryotic mRNA are polygenic
• Poly-A tail – the 3’ end most eukaryotic
  mRNAs have a ‘tail’ of 100 – 200
  successive A residues
Intron RNA must be removed from
        mRNA precursor
Small nuclear RNAs aid in removing intron RNA
• Small nuclear RNAs aid in removing intron
  RNA
• The transcription process can be visualized
• DNA is transcribed from some viral RNAs
  by reverse transcriptase
• Some viral RNAs are replicated by an RNA
  directed RNA polymerase
• Polynucleotide phosphorylase makes
  random RNA-like polymers

								
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