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DNA REPLICATION - DOC

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					BIO 113 W05 Lecture Outline: DNA REPLICATION, RNA Synthesis, Protein Synthesis

I    INTRODUCTION TO DNA REPLICATION AND DNA STRUCTURE
A    What is DNA Replication?




B    Where Does DNA Replication Occur?

C    Nucleic Acids are made up of Nucleotides                     OH fig 2.46
     1     structure of a nucleotide
           a      5C sugar:

           b     nitrogenous base: Guanine, Adenine, Cytosine, Thymine, Uracil
                 1     purine: 2 rings, A, G
                 2     pyrimidine: 1 ring, CTU

           c     phosphate group

     2     Nucleotides on the same strand are connected by phosphodiester bonds
           a    5' end =

           b     3' end =

           c     the bonds between sugars and P form the sugar phosphate backbone
                  5'P-nt1-OH * P-nt2-OH * P-nt3-OH

           d     DNA is always synthesized in the 5' to 3' direction

D    DNA = deoxyribonucleic acid                            OH fig 16-4b
     1   function:
     2   structure:
         a      double stranded=ds, double helix
         b      strands of DNA are antiparallel

           c     nucleotides on opposite strands are connected by H bonds between
                 complementary bases = base pairs
                 1     A::T in DNA, A::U in RNA
                 2     G:::C
                                                                                    1
II    DNA SYNTHESIS: GENERAL
A     DNA Replication is semiconservative.


B     DNA replication is bidirectional.
      DNA opens up in areas called origins of replication (ORI), in middle of chromosome
      and proceeds towards both ends

III   ENZYMES INVOLVED IN DNA SYNTHESIS (PROKARYOTES)
A     DNA Polymerase:

      1     DNA Polymerase III:
            a   5' -> 3' polymerase activity
            b   3' -> 5' exonuclease activity

      2     DNA Polymerase I:

B     Primase:

C     Helicase: binds to ssDNA, unwinds dsDNA

D     Topoisomerase:

IV    STEPS IN DNA REPLICATION

A     step 1:
      enzymes:

B     step 2:
      enzyme:
      primer:

C     step 3: DNA is synthesized in the 5' -> 3' direction
      1     enzyme:

      2     DNA is synthesized on the leading strand as a continuous strand (5' -> 3')

      3     DNA is synthesized on the lagging strand as short DNA sequences (5' -> 3')
            Okazaki fragments:

      4     Proofreading:

                                                                                           2
D    step 4:
     enzyme:

E    step 5:
     enzyme:

F    Result of DNA Replication:

CD Rom: DNA Replication

V    CENTRAL DOGMA: DNA -> RNA -> PROTEIN
A    DNA Is Arranged Into Genes
     1   gene:


     2    to be expressed, a gene must be:
          a      transcribed into
          b      translated
          c      the protein must be

     3    gene structure
          a     promoter:


          b     coding region:

                1     contains start and stop sites for RNA synthesis = transcription
                2     exons
                3     introns

     4    one gene, one enzyme:


B    DNA is transcribed into RNA
     1    transcription:

     2    enzyme involved:

     3    location:

     4    purpose:


                                                                                        3
          Reminder: there are three main types of RNA in the cell
          a    mRNA:
          b    rRNA:
          c    tRNA:

5   When does this occur in the cell cycle?


C   Steps in Transcription
    1     RNA Polymerase binds to the


    2     RNA Polymerase starts making a new RNA molecule, using DNA as a template
          a   RNA is always made from 5' to 3'

          b         during transcription a new RNA is made from the DNA template by
                    adding the complementary base into the new RNA
                    DNA A= U RNA, DNA G= C RNA, DNA C= G RNA, DNA T= A RNA

          c         if template strand of DNA is:   3’-AAT GCA TGT CGA-5’
                    then the RNA sequence is:       5’-UUA CGU ACT GCU-3’

    3     a cap

    4     when a termination signal is reached, transcription stops and a polyA tail is added
          termination signal = sequence in DNA, makes RNA synthesis stop
          termination signal NOT the same as STOP CODON

    5     at the end of transcription we have a pre-mRNA

          cap – exon intron exon – AAAAAAA              =nonfunctional pre-mRNA

    6      Next step = RNA Splicing.

    7     result:

D   mRNA is Translated into Protein
    1  translation:

    2     location:

    3     when does translation occur in the cell cycle?

                                                                                            4
    4     molecules involved in protein synthesis:
          a    ribosome:

          b         amino acids:

          c         mRNA:
                    contains codons =

          d         tRNA:
                    anticodon

          e         universal code: every codon = three nucleotides codes for an amino acid
                    or a stop signal
                    1      each codon is specific: it codes for 1 and only one amino acid

                    2     the universal code is redundant: more than one codon can code for
                          a particular AA

                    3     each codon on the mRNA is recognized by base-pairing with an
                          anticodon on a tRNA= triplet of nucleotides


    4     result:


E   Steps in Translation: Initiation
    1     small subunit of ribosome binds to the mRNA
          the small subunit has two positions where tRNAs can bind
          a     P site = 1st position

          b         A site = 2nd position

    2     first tRNA binds to the first position = P site of small subunit

    3     large subunit of ribosome binds

F   Steps in Translation: Chain Elongation                            0
    1     1st tRNA is already in the P site= 1st position

    2     2nd tRNA binds to the A site=2nd position

    3     a peptide bond is formed between

                                                                                              5
      4    the AA is transferred from the first tRNA to the AA in the A site

      5    the first tRNA (now lacking an AA) leaves the P site

      6    ribosome moves forward one position
           a     AA1-AA2-tRNA are now in the P site

           b     the A site is empty and ready for a new tRNA-AA

      7    these steps are REPEATed until a STOP codon is reached


G     Steps in Translation: Termination
      1     ribosome comes to a STOP codon (STOP codon in the A site)

      2    a release factor binds to the STOP codon

      3    the release factor breaks the bond between the tRNA and the polypeptide

      4    the polypeptide is released from the tRNA in the P site

      5    the tRNA is released from the ribosome, it will be recharged with another AA

      6    the large and small subunits separate, they can be reused to translate another
           mRNA

      7    the mRNA is released, it can be reused

MUTATIONS

I     What is a Mutation?


II    What causes mutations?
      A    although DNA replication is very accurate sometimes mistakes occur
           sometimes proofreading does not detect all mismatched bases

      B    chemicals

      C    UV light

           Xeroderma pigmentosa: defect in DNA repair enzymes that remove thymine
           dimers
III   Types of Mutations
                                                                                            6
A    Point Mutation:
     1     silent mutation:

           Result:

           DNA         CCG->        CCA
           RNA         GGC->        GGU
           AA          Gly          Gly

     2     missense mutation:

           Result:

           DNA         CCG->        TCG
           RNA         GGC ->       AGC
           AA          Gly          Ser

     3     nonsense mutation:

           Result:

           DNA         TTC->        ATC
           RNA         AAG->        UAG
           AA          Lys          stop

B    Frameshift mutation:
     1   since the mRNA is read as a triplet (3 nt = codon), any change in the number of
         nt will change the reading frame of the mRNA

     2     all of the codons from the inserted/ deleted nt onwards will be changed

     3     Result:

Original DNA     3’-   TAC TTC AAA CCG ATT              TGTAACT-5’
Original mRNA    5'-   AUG AAG UUU GGC UAA             ACAUUGA-3'
                       MET Lys Phe Gly Stop

Mutant DNA       3’-   TAC TTC AA C CGA TTT GTA ACT-5’

Mutant mRNA      5'-   AUG AAG UUG GCU AAA CAU UGA-3'
                       MET Lys Leu  Ala Lys His Stop


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