Protein Synthesis DNA at work

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)

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)

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?

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

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

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”

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

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

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

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

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

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

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

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

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

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

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

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

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

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

DNA – RNA - Protein Gene expression

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

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?

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

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)

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)