1. Protein Synthesis
DNA at work

2. If DNA = recipe book Proteins = courses of a meal
Recipes for all polypeptides are encoded by DNA
mRNA is a copy of that recipe (DNA sequence)
mRNA (recipes) travel to ribosomes for translation into polypeptides (proteins)

3. Early developments
1909: A. Garrod suggests that “genes” create phenotypes via enzymes
Genes: heritable units of DNA
Phenotype: observable characteristic
People who lack particular enzymes have disease phenotypes (metabolic incompetence)

4. Early developments
1940’s: Beadle & Tatum; Neurospora crassa (mold) produce thousands of offspring; some cannot grow on traditional food source = nutritional mutants
Could these mutants lack an enzyme?

5. Early developments They do!
It’s often one dysfunctional enzyme per mutant, and one dysfunctional gene
One gene-one enzyme hypothesis
One gene-one protein
One protein-one polypeptide

6. Protein recipe is written in genetic code (genes)
Genes lie along DNA
What are chromosomes?
Genes are linear sequences of nucleotides
One, three-nucleotide sequence = codon

7. Genetic code & codons Each codon codes for a particular Amino Acid
Each gene has many codons in it
Codons also exist for “start translating” and “stop translating”

8. Genetic code & codons Redundant – multiple codons specify same AA
Unambiguous - NO codon specifies more than one AA
Ancient – ALL organisms have same genetic code
AUG = Methionine whether you’re a redwood or a fruitfly

9. How RNA is made RNA polymerase adds RNA nucleotides to DNA template
RNA molecule peels away from DNA strand

10. How RNA is made
Initiation: RNA polymerase binds to a promoter (specific nucleotide sequence)
Elongation: Polymerase adds complementary nucleotides to DNA template; RNA peels away, DNA reconnects

11. How RNA is made
Termination: RNA polymerase reaches “terminator sequence”.
RNA polymerase detaches; mRNA detaches

12. Further processing
Addition of caps (G) & tails (poly A) by RNA polymerase
Allow recognition by ribosomes (Cap, Tail)
Protect RNA from RNase attack (Cap)
Protect RNA from exonuclease attack (Tail)
Allow export by transporter molecules

13. Further processing Introns spliced out Exons joined
Intervening sequences; NOT transcribed into polypeptide
Exons joined
Coding regions of DNA that are transcribed into Amino Acids

14. tRNA brings appropriate AA
tRNA is “cook’s helper”
Brings individual ingredients (AA) to make the recipe (protein)
Binds appropriate AA (in cytoplasm)
Recognizes the mRNA codon that specifies its AA
Complementary nucleotide sequence (Anticodon) for recognition

15. tRNA binding sites
Anticodons & AA attachment sites are themselves a string of three nucleotides
One enzyme attaches each AA to any of its possible tRNA transporters

16. Ribosomes & Translation
rRNA plus proteins
2 rRNA subunits
Bind mRNA
Bind tRNA with attached Amino Acids

17. Ribosomes Small subunit binds mRNA
Large subunit, with tRNA binding sites, attaches to small subunit + mRNA

18. Translation Initiation mRNA binds to small subunit.
Initiator tRNA binds to start codon, always AUG -> first AA of all polypeptides is always Met

19. Translation* Elongation
Large subunit binds to small -> functional ribosome
Initiator tRNA attaches to P site of ribosome. Holds growing polypeptide. Next tRNA attaches to A site

20. Translation Elongation
Codon recognition: tRNA anticodon binds to mRNA codon in the A site
Peptide bond formation: Polypeptide detaches from tRNA in P site & binds to AA & tRNA in A site

21. Translation Elongation Termination
Translocation: tRNA in P site detaches, A site tRNA & mRNA move, as unit, into P site. New tRNA attaches to A site.
Termination
Stop codon is reached; no AA is added; polypeptide releases & subunits dissociate

22. DNA – RNA - Protein
Gene expression

23. Mutations Any change in nucleotide sequence
Substitutions
Insertions
Deletions
Many alternative phenotypes result from single nucleotide changes

24. Point Mutations Substitution: A single base pair is changed.
Synonymous (silent): results in NO AA change…why not?
Nonsynonymous: results in single AA change
These are less likely to be deleterious. WHY?

25. Example* Hemoglobin mutations
HbE: Codon position 26; Replace GLU w/ LYS; reduced Hb production. Hemoglobin instability at low O2
HbC: Position 6; Replace GLU w/ LYS; RBC’s become rigid & crystalize
HbS: Position 6; Replace GLU w/ VAL; At low O2, Hb polymerizes & RBC’s collapse

26. Point Mutations Indels: insertions/ deletions
A single nucleotide is inserted or deleted
Far more likely to be deleterious because these shift the reading frame (triplet grouping)

27. Sources of mutation* Mutagenesis: Production of mutations
Spontaneous mutations:
Errors in replication coupled with subsequent errors in proofreading
Errors in chromosome (DNA) separation during cell division
Mutagens: Physical or chemical agents
X-rays, UV light (high energy photons)