Protein Synthesis

DNA at work

If DNA = recipe bookProteins = 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

1. Initiation: RNA polymerase binds to a promoter (specific nucleotide sequence)

2. Elongation: Polymerase adds complementary nucleotides to DNA template; RNA peels away, DNA reconnects

How RNA is made

3. Termination: RNA polymerase reaches “terminator sequence”.

3. 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

– 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

Translation1. Initiation

• mRNA binds to small subunit.• Initiator tRNA binds to start codon, always

AUG -> first AA of all polypeptides is always Met

Translation*2. 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

Translation2. Elongation

1. Codon recognition: tRNA anticodon binds to mRNA codon in the A site

2. Peptide bond formation: Polypeptide detaches from tRNA in P site & binds to AA & tRNA in A site

Translation2. Elongation

3. Translocation: tRNA in P site detaches, A site tRNA & mRNA move, as unit, into P site. New tRNA attaches to A site.

3. 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)