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
 Learning Objectives
• Understand the structure of the ribosome and polysome
• Understand the role of tRNA as an "adaptor"
• Understand the mechanism for charging tRNAs
• Understand the general characteristics of the genetic code
• Know the start and stop codons
• Understand the mechanism for how some tRNAs can recognize more
than one codon
• Understand how the AUG initiation codon is recognized in prokaryotes
and eukaryotes
• Understand how the AUG codon establishes the reading frame
• Know the various kinds of mutations that affect the translation of
proteins
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
Messenger RNA (mRNA)

initiation
Cap codon
5’ untranslated region
5’ m7Gppp AUG

translated (coding) region

UGA
termination
3’ untranslated region codon

AAUAAA (AAAA)n 3’
poly(A)
tail
Ribosomes
• prokaryotic ribosome

50S subunit 70S ribosome 30S subunit

23S rRNA 16S rRNA
5S rRNA 21 proteins
35 proteins

• eukaryotic ribosome

60S subunit 80S ribosome 40S subunit

28S rRNA 18S rRNA
5S rRNA 33 proteins
5.8S rRNA
49 proteins
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

5’ AUG UGA

polysome

small ribosomal subunit subunits dissociate

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
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
 amino acid
uncharged tRNA
RO

=
- -
H2N-C-C-OH 3’
H
ATP
adenylated (activated)
RO amino acid
=
- -

H2N-C-C-O-P-O-ribose-adenine
PPi
H

AMP
RO

=
- -
H2N-C-C-O
Amino acid activation H
and
tRNA charging aminoacyl
(charged)
tRNA
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

• 3 additional amino acids (Arg, Leu, and Ser) are specified by

six different codons
 The Genetic Code

UUU UCU UAU UGU

Phe Tyr Cys
UUC UCC UAC UGC
Ser
UUA UCA UAA UGA Stop
Leu Stop
UUG UCG UAG UGG Trp

CUU CCU CAU CGU

His
CUC CCC CAC CGC
Leu Pro Arg
CUA CCA CAA CGA
Gln
CUG CCG CAG CGG

AUU ACU AAU AGU

Asn Ser
AUC Ile ACC AAC AGC
Thr
AUA ACA AAA AGA
Lys Arg
AUG Met ACG AAG AGG

GUU GCU GAU GGU

Asp
GUC GCC GAC GGC
Val Ala Gly
GUA GCA GAA GGA
Glu
GUG GCG GAG GGG
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 3’ 5’ tRNAmet
• G with C or U
• U with A, G, or I
• I with C, U, or A UAC
AUG
mRNA
5’ 3’
3’ 5’ tRNAleu
• one tRNAleu can read two
of the leucine codons
GAU wobble base

CUA
mRNA G
5’ 3’
 Wobble Interactions

Inosine = Cytidine Inosine = Adenosine

Inosine = Uridine Guanosine = Uridine

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
lac I P O lac Z lac Y lac A
AUG AUG AUG AUG

5’ SD AUG AUG SD AUG

initiation codon with internal Met codon initiation codon with
Shine-Dalgarno site does not have Shine-Dalgarno site
Shine-Dalgarno site

• eukaryotic mRNA
5’ cap AUG AUG
internal (downstream) Met codon
initiation can only occur at cannot serve as an initiation site
first AUG codon downstream of the 5’ cap
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.UCGAAUAAA...
MET.ALA.GLU.TRP.LEU.SER.MET.SER

• a frameshift mutation

...AGAGCGGA.AUG.GCA.GA .UGG.CUA.AGC.AUG.UCG.UGA.UCGAAUAAA...

• the new reading frame results in the wrong amino acid sequence and
the formation of a truncated protein

...AGAGCGGA.AUG.GCA.GAU.GGC.UAA.GCAUGUCGUGAUCGAAUAAA...
MET.ALA.ASP.GLY
Mutations affecting translation
• hemoglobin Wayne (3’ terminal frameshift mutation)

Normal α -globin .ACG.UCU.AAA.UAC.CGU.UAA.GCU GGA GCC UCG GUA

.THR.SER.LYS.TYR.ARG

Wayne α -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