Transcript Protein Synthesis & Mutation

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
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
1. Initiation
•
•
Translation
mRNA binds to small subunit.
Initiator tRNA binds to start codon, always AUG ->
first AA of all polypeptides is always Met
2. Elongation
•
•
Translation*
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
2. 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
Translation
2. 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)