7' Protein Synthesis and the Genetic Code

1
7. Protein Synthesis and the Genetic Code
a). Overview of translation i). Requirements
for protein synthesis ii). messenger
RNA iii). Ribosomes and polysomes iv).
Polarity of protein synthesis b). Transfer
RNA i). tRNA as an adaptor ii). Amino acid
activation iii). Aminoacyl tRNA
synthetases iv). Charged tRNA c). The
genetic code i). Codon-anticodon
interactions ii). Initiation codon in
prokaryotes vs. eukaryotes iii). Reading
frame d). Mutations affecting translation i).
Frameshift mutations ii). Missense and nonsense
mutations
2

• Overview of translation
• last step in the flow of genetic information
• definition of translation
• requirements for protein synthesis
• mRNA
• ribosomes
• initiation factors
• elongation and termination factors
• GTP
• aminoacyl tRNAs
• amino acids
• aminoacyl tRNA synthetases
• ATP

3
Messenger RNA (mRNA)
initiation codon
Cap
5 untranslated region
5
AUG
m7Gppp
translated (coding) region
UGA
termination codon
3 untranslated region
(AAAA)n
3
AAUAAA
poly(A) tail
4

• Ribosomes
• prokaryotic ribosome
• eukaryotic ribosome

5

• Polysomes
• direction of translation is 5 to 3 along the
  mRNA
• direction of protein synthesis is N terminus to
  C terminus

nascent polypeptide
large ribosomal subunit
N
N
UGA
5
AUG
polysome
small ribosomal subunit
subunits dissociate
6

• Transfer RNA
• tRNA is the adaptor molecule in protein
  synthesis
• acceptor stem
• CCA-3 terminus to which amino acid is coupled
• carries amino acid on terminal adenosine
• anticodon stem and anticodon loop

7

• Amino acid activation and aminoacyl tRNA
  synthetases
• aminoacyl tRNA synthetases are the enzymes that
  charge the tRNAs
• 20 amino acids
• one aminoacyl tRNA synthetase for each amino
  acid
• can be several different isoacceptor tRNAs for
  each amino acid
• all isoacceptor tRNAs for an amino acid use the
  same synthetase
• each aminoacyl tRNA synthetase binds
• amino acid
• ATP
• isoacceptor tRNAs

8
amino acid
uncharged tRNA
3
ATP
adenylated (activated) amino acid
PPi
AMP
Amino acid activation and tRNA charging
aminoacyl (charged) tRNA
9

• The genetic code
• consists of 64 triplet codons (A, G, C, U) 43
  64
• all codons are used in protein synthesis
• 20 amino acids
• 3 termination (stop) codons UAA, UAG, UGA
• AUG (methionine) is the start codon (also used
  internally)
• multiple codons for a single amino acid
  degeneracy
• 5 amino acids are specified by the first two
  nucleotides only

10
The Genetic Code
UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG
GUU GUC GUA GUG
UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG
GCU GCC GCA GCG
UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG
GAU GAC GAA GAG
UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG
GGU GGC GGA GGG
Ser Pro Thr Ala
Tyr Stop His Gln Asn Lys Asp Glu
Cys Arg Ser Arg Gly
Phe Leu Leu Val
Stop Trp
Ile Met
11

• Codon-anticodon interactions
• codon-anticodon base-pairing is antiparallel
• the third position in the codon is frequently
  degenerate
• one tRNA can interact with more than one codon
  (therefore 50 tRNAs)
• wobble rules
• C with G or I (inosine)
• A with U or I
• G with C or U
• U with A, G, or I
• I with C, U, or A

3
5
tRNAmet
U A C
A U G
mRNA
5
3
3
5
tRNAleu

• one tRNAleu can read two
• of the leucine codons

wobble base
G A U
C U A G
mRNA
5
3
12
Wobble Interactions
13

• Initiation in prokaryotes and eukaryotes
• initiation can occur at internal AUG codons in
  prokaryotic mRNA
• initiation in eukaryotes occurs only at the
  first AUG codon
• lac operon in E. coli is transcribed as a
  polycistronic mRNA
• with multiple AUG codons
• eukaryotic mRNA

lac I
P
O
lac Z
lac Y
lac A
AUG
AUG
AUG
AUG
5
AUG
SD
AUG
SD
AUG
initiation codon with Shine-Dalgarno site
initiation codon with Shine-Dalgarno site
internal Met codon does not have Shine-Dalgarno
site
5 cap
AUG
AUG
internal (downstream) Met codon cannot serve
as an initiation site
initiation can only occur at first AUG codon
downstream of the 5 cap
14

• Reading frame
• reading frame is determined by the AUG
  initiation codon
• every subsequent triplet is read as a codon
  until reaching a stop codon

• ...AGAGCGGA.AUG.GCA.GAG.UGG.CUA.AGC.AUG.UCG.UGA.UC
  GAAUAAA...
• MET.ALA.GLU.TRP.LEU.SER.MET.SER
• a frameshift mutation
• ...AGAGCGGA.AUG.GCA.GA .UGG.CUA.AGC.AUG.UCG.UGA.UC
  GAAUAAA...
• the new reading frame results in the wrong amino
  acid sequence and
• the formation of a truncated protein
• ...AGAGCGGA.AUG.GCA.GAU.GGC.UAA.GCAUGUCGUGAUCGAAUA
  AA...
• MET.ALA.ASP.GLY

15

• Mutations affecting translation
• hemoglobin Wayne (3 terminal frameshift
  mutation)

Normal a-globin .ACG.UCU.AAA.UAC.CGU.UAA.GCU GGA
GCC UCG GUA .THR.SER.LYS.TYR.ARG Wayne
a-globin .ACG.UCA.AAU.ACC.GUU.AAG.CUG.GAG.CCU.CGG.
UAG .THR.SER.ASN.THR.VAL.LYS.LEU.GLU.PRO.ARG
mutated region

• missense mutations (e.g., AGC Ser to AGA Arg)
• nonsense mutations (e.g., UGG Trp to UGA Stop)
• read through, reverse terminator, or sense
  mutations
• (e.g., UAA Stop to CAA Gln) as in hemoglobin
  Constant Spring
• silent mutations (e.g., CUA Leu to CUG Leu) do
  not affect translation