I.  DNA Transcription (RNA Synthesis)

   A. Introduction

•  RNA is synthesized using DNA as a template
  
  
•  Three primary types of RNA
   

   B. Transcription in procaryotes

        1. Gene

•   polycystronic (polygenic) mRNA
  
  
•   transcription of anti-sense strand
   

         2. Initiation

•  RNA polymerase, five subunits
  
  
• Sigma subunit determines binding of RNA polymerase to DNA promoter.
  
  
• transcription start point
   
   

         3. Elongation

•  sigma released
  
  
• RNA polymerase moves along DNA strand
   

         4. Termination

•  Termination (stop signal) tells RNA polymerase to stop adding nucleotides.
  
   

  5.  Regulation

     a)  inducible vs. constitutive

     b)  promoters

     c)  repressor (negative control)

     d)  activator (positive control)

     e)  summary

• rate of transcription and stability of mRNA is relatively constant
  
  
• DNA replication and transcription occur simultaneously

          6. Transcription inhibitors

•  Actinomycin D prevents RNA polymerase from moving along DNA.
  
  
•  Rifamycins (e.g. rifampin) inhibits transition from initiation to elongation.
   
   

      C.  Transcription in eucaryotes

          1. RNA polymerases

                RNA polymerase I

                RNA polymerase II

                RNA polymerase III: tRNA
 
 

         2.  Posttranscriptional modification

•  heterogeneous nuclear RNA (hnRNA)
  
  
• poly-A (adenylate) tail
  
  
• 5' cap of 7-methylguanosine
  
  
• Exons and introns
  
  
• RNA splicing
   

II. Protein Synthesis (Translation)

   A.  tRNA

•  Carries specific amino acid corresponding to anti-codon triplet.
  
  
• Activated by aminoacyl-tRNA synthetase to aminoacyl-tRNA
   
   

   B.  Ribosomes

        differences between procaryote and eucaryote
 
 

   C.  Initiation of protein synthesis

•   N-formylmethionyl-tRNAfmet (fmet-tRNA) binds to free 30s subunit.
  
  
•   mRNA
  
  
• 50s subunit
   

   D.  Elongation of polypeptide chain

            Amino acids linked together by transpeptidation reaction.
 

   E.  Termination of protein synthesis

            nonsense (stop) codon (UAA, UAG, UGA)
 

   F.  Summary

•   In procaryotes, transcription and translation occur simultaneously.
  
  
•   Polyribosomes (polysomes)
  
  
•   Protein synthesis rather energy expensive.
  
  
•  Inhibitors of protein synthesis: aminoglycosides (streptomycin, kanamycin, gentamycin), and tetracyclines
  
  
•  Post-translational modification
   

II.  Recombination and Gene Transfer

•  Recombination: one or more nucleic acid molecules rearranged or combined to produce a new nucleotide sequence

    B.  Gene Transfer

• three mechanisms of gene transfer: conjugation, transformation, and transduction
  
  
• difficult getting DNA across plasma membrane

•  replicate independent of the chromosome
  
  
• R factors (plasmids)
   
   

    D.  Transposable Elements

• Pieces of DNA that move around the genome–process referred to as transposition
  
  
• Insertion sequence (IS) is the simplest transposable element.
  
  
• composite transposons

    E.  Bacterial Conjugation

•   Exchange of DNA (genetic information) by direct cell contact

    F.  DNA Transformation

•  Griffith's experiment
  
  
•  Research technique
   

        2. Specialized transduction
 

III. Recombinant DNA Technology

    A. Introduction

•   Use of recombinant DNA technology to deliberately modify an organism's genetic make up (genetic   engineering)
  
  
•   Involves many techniques
  
  
•   Restriction enzymes
  
  
•   Cohen and Boyer (1973), inserted antibiotic resistant gene into E. coli
  
  
•   Polymerase chain reaction (PCR), Mullis (1983)
   

        1. Medical applications and research

• restriction fragment length polymorphism (RFLP, 1978)
  
  
• production of human protein
  
  
• gene therapy (1990)
  
  
• recombinant vaccines
  
  
• forensics and paternity testing
  
  
• ANDi
   
   

        2. Industrial

• Bioremediation
   
   

        3. Agricultural

• Flavr Savr tomatoes (1994)
  
  
• StarLink's genetically modified corn (1998)
  
  • Bt corn
     

4. Cloning animals


• more accurately, nuclear transfer
  
  
• Dolly the sheep (1997)
  
  
• CC (Copy Cat, 2001)

 

    C. Summary

• human genome project
  
    
• Social and moral issues--how far should scientists go?
  
  
• gene therapy
  
  
• humanizing pig genes
  
  
• therapeutic cloning