DNA Replication & Transcription


In principle: DNA replication is semi-conservative
       H - bonds 'unzip', strands unwind,
        complementary nucleotides added to existing strands
             After replication, each double-helix has one "old" & one "new" strand
                  [note alternative conservative & dispersive models:  Homework #4 ]

       DNA is not  the "Genetic Code" for proteins
               information in DNA must first be transcribed into RNA
               messenger RNA transcript is base-complementary to template strand of DNA
                                                                       & therefore co-linear with sense strand of DNA

       DNA & RNA syntheses occur only in the  5'  3' direction

Central Dogma



DNA synthesis in prokaryotes:
     Nucleotides are added simultaneously to both strands, but
     DNA grows in the 5'  3' direction ONLY  [iG1 10.10]

         (online animation)

 Distinguish:
   Replication: duplication of a double-stranded DNA (dsDNA) molecule
                         an exact 'copy' of the existing molecule (cf. xerox copy)
   Synthesis: biochemical creation of a new single-stranded DNA (ssdNA) molecule
                        a base-complementary 'copy' of an existing strand (cf. silly putty copy)
                        occurs only in the 
5'  3' direction

   Homework #5

   DNA Synthesis in prokaryotes 
         Formation of replication fork at Origin of Replication [iG1 10.16, 19]
                   provides two single-stranded DNA template (ssDNA)
                  multiple replications forks (
replicons)
        Synthesis of RNA primer [iG1 10.15]
 
       Addition of dNTPs  by  DNAPol III  at 3' end only
                  continuous synthesis on leading strand [iG1 10.13]
                  discontinuous synthesis on lagging strand [iG1 10.20]
                     Okazaki fragments
                        proof-reading  by 3'5' exonuclease activity [iG1 10.12]
                  
leading & lagging strand synthesis simultaneously [iG1 10.21]
                       A single, dimeric DNAPol III replicates both strands
        Excision of RNA primer by DNAPol I

              ligation
(connection) of fragment ends at gaps by DNA ligase  [iG1 10.22]

              A talkie animation of DNA synthesis `[onlineMGA2 animation]

    DNA synthesis in eukaryotes

       Eukaryotic genomes are much larger [the "C-value Paradox"]
                  eukaryotic DNA synthesis is more efficient:
       More DNAPol molecules, slower rate of synthesis, more replicons,


Transcription: synthesis of messenger RNA (mRNA) (online MGA2 animation)


    What is a "Gene" [iGen3 05-03]


     RNA transcribed from DNA by RNA Polymerase (RNAPol I)  [iG1 4.17]
            (1) Recognition of transcriptional unit: ~ 'gene' [iG1 4.18]
                      Promoters - short DNA sequences that regulate transcription
                          typically 'upstream' = 'leftward' from 5' end of sense strand [iG1 4.12]
            (2) Initiation & Elongation [iG1 4.22, 23, 24]
                      mRNA synthesized 5'3'  from DNA template strand
                      mRNA sequence therefore homologous to DNA sense strand

                          Colinear: mRNA and DNA sense strand "line up"

                                            (in prokaryotes, but not eukaryotes: see below)
                          Process similar to DNA replication [iG1 4.25], except
                               No primer
is required

                              Transcription may occur from either strand

                               Most DNA is not transcribed into RNA

            (3) Termination [iG1 4.27, 28, 29]

    Regulation of transcription
          In prokaryotes, transcription & translation may occur simultaneously
          In eukaryotes, transcription occurs in nucleus [ex.: Lampbrush chromosomes]
                                     translation occurs in cytoplasm (see next section):
              RNA must cross nuclear membrane
                        transcription  & translation are physically separated
                        primary RNA transcript is extensively processed
                        heterogeneous nuclear RNA (hnRNA mRNA

    Post-transcriptional processing of eukaryotic RNA is complex [iG1 4.9]
          promoters  [iG1 4.19] & enhancers [iG1 ] determine initiation & control rate
          'cap' (7-methyl guanosine, 7mG) added to 5' end  [iG1 4.26]
          'tail' of poly-A (5'-~~~AAAAAAAAAA-3') added to 3' end [iG1 4.33]
          'splicing' of hnRNA : eukaryotic genes are "split"
              intron DNA sequence equivalents removed from hnRNA : "intervening"
              exon   DNA sequence equivalents represented in mRNA"expressed" in protein
                        1 ~ 12's of exons / 'gene'
                         >90% of transcript may be 'spliced out'
                              [An important note on terminology]
             Splicing mechanism uses donor and acceptor sites [iG1 5.18, 19, 20]

             Eukaryotic genes & mRNA are not colinear!
                DNA / RNA hybridization produces heteroduplexes
                    DNA introns 'loop out'
                    DNA exons pair with mRNA
                 Eukaryotic exons may be widely separated
    
     Alternative splicing of the same transcript produces different products [iG1 4.16]
        Different exon regions are combined as different mRNAs [iG1 5.01]
        Alternative exon combinations differ functionally [iG1 5.22]

   Summaries of transcription [& translation] in prokaryotes & eukaryotes



Homework #6: Problem-solving with DNA & RNA

Ongoing Homework problem:
       What is a 'gene'? How do the discoveries of (1) introns and exons amd (2) alternative splicing in eukaryotic genomes modify the concept?


All text material 2013 by Steven M. Carr