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Biochemistry I, Spring Term
Lecture 39
May 1, 2013
Lecture 39: tRNA Charging, Codon-anticodon Interactions, Intro. to Protein Syn.
RNA molecules involved in protein synthesis:
a) mRNA – messenger RNA is copy of the DNA that encodes a gene. mRNA specifies the order of amino acids
to be used in making the protein.
b) tRNA – transfer RNA is the dictionary the converts the codon to a specific amino acid. One part of the tRNA
recognizes the codon, the other part contains the aminoacid to add.
c) rRNA – ribosomal RNA is found in the ribosome and is responsible for most of the function in protein
synthesis.
tRNA: Although it varies, there are generally 25-45 different tRNAs/organism. This complex single chain RNA
molecule structure is stabilized by W-C H-bonds, non-W-C H-bonds, and phosphate-metal interactions.
 Acceptor stem: amino acids are attached to the 3' terminus of the tRNA by enzymes called aminoacyltRNA Synthetases (aaRS). These enzymes attach the correct amino acid to the correct tRNA. There are
~25-30 of these enzymes, essentially one for each amino acid. This process is often referred to as
“charging” the tRNA.
 Anti-codon arm: contains the anticodon triplet that translates the codon in mRNA to an amino acid. WatsonCrick H-bonds are used here.
"Charging" of tRNA: amino acyl tRNA synthetases.
tRNA Charging: [AAX + ATP → AAX-AMP + 2 Pi] + tRNAX → tRNAx-AAX + (AMP +2Pi)
1. Activation of Amino acids (2 ATP equivalents)
+
H3N
O
O
O
O
O
O P O P O P O
O
O
O
O
+
H3N
O
Adenosine
O
O P O
O
O
Adenosine
ATP
OHOH
1
OHOH
+
O
O
O P O P O
O
O
pyrophosphate
2x O
O P O
O
Biochemistry I, Spring Term
Lecture 39
May 1, 2013
2. Transfer to tRNA
tRNA
tRNA
O O
P O
O
Cytosine
O
OH
O O
P O
O
AAG
O O
P O
O
O
O P
H3N
AAG
OH
O
O
O P
+
O
O
Adenosine
O
O
O
+
Cytosine
OH
O O
P O
O
Adenosine
OH OH
O
Phe
O
O
NH3
Adenosine
O
O
O
OH OH
OH OH
2 x Pi
ATP/AMP site
G<<0
AA site
H2 N
O
O
H2 N
P-P
O
O
ATP
AMP
Phe
ATP
tRNA site
H2 N
amino-acyl tRNA
synthetase (Phe)
3'
AAG
H2 N
Adenosine
H2 N
O
O
AMP
O
O
H2 N
AMP
AAG
O
O
O
O
+
"Charged"
tRNA
AMP
AAG
AAG
AAG
5' Base
Codon-Anticodon Interactions
U (=T)
1. Charged tRNAs are selected by ribosomes solely through codonanticodon interactions.
2. Degeneracy at the third position of codon-anticodon
pairing allows multiple codons/tRNA.
Example: pairing combinations for tRNAPhe
(superscript ‘Phe’ indicates that this tRNA will be
attached to Phenylalanine.)
i) The codon-anticodon pairing is anti-parallel, as
are most pairings of nucleic acid strands:a)
ii) The anticodon on the tRNA is 5'-GAA-3'.
iii) The complementary codons in the mRNA are 5'UUC-3' and 5'-UUU-3'.
iv) The anticodon GAA can pair with either codon
due to degeneracy at the third position (wobble
basepair).
H2 N
N
H
H2 N
O
O
AAG
5'---UUU----3'
N
N
H
N
Rib N
N
N
N
O
H
O
H
O
H
N
H
N
N
H
C-G 'Watson-Crick' Base Pair
U
C
A
G
Rib
H
N
3'
O
O
AAG
O
2
Middle Base
C
A
G
Ser
Tyr
Cys
Ser
Tyr
Cys
Ser Term Term
Ser Term
Trp
5'---UUC----3'
Rib
N
N
H
U
Phe
Phe
Leu
Leu
Rib
N
O
U-G Wobble Base Pair
Biochemistry I, Spring Term
Lecture 39
May 1, 2013
Protein Synthesis - Overview:
Leader peptide
(export)
1. The information content of the mRNA is translated into
a polypeptide chain by the ribosome. Three nucleotide
bases, or a codon, encode each amino acid.
2. mRNA is the template, and synthesis of the polypeptide
chain proceeds in the aminocarboxy direction as
new amino acids are added to the carboxy terminus of
the growing peptide chain.
Start codon
Ribosome binding
site
HIV Protease
Coding Region
His-Tag
(purification)
Stop codon
EcoR1
Lac Operator
mRNA
Termination
BamH1
Promoter
Antibiotic
Resistance
Gene
A. Features of the mRNA (Example - Synthesis of Met-Lys-Ala).
Origin of
Replication
Beginning with the DNA:
TTGACATTTATGCTTCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAGGAACAGCTATGAAAGCTTAATTTATG.
AACTGTAAATACGAAGGCCGAGCATATTACACACCTTAACACTCGCCTATTGTTCCCGTGTGTCCTCCTTGTCGATACTTTCGAATTAAATAC.
-35
-10
→
Lac operator
Promoter
mRNA start
The mRNA:
Without punctuation:
GAAUUGUGAGCGGAUAACAAUUUCACACAGGAGGAACAGCUAUGAAAGCUUAAUUUAUG.....
With punctuation
GAAUUGUGAGCGGAUAACAAUUUCACACAGGAGGAACAGCUAUG,AAA,GCU,UAA,UUU,AUG...
fMet-Lys-Ala-STOP
Ribosome Binding Site (RBS):
(Shine-Dalgarno [SD] sequenceAGGAGG.)
Positions mRNA on the ribosome
so that the correct start codon is
used.
Complementary to 16s rRNA in 30s
subunit.
The optimal spacing between the
SD sequence and the AUG is 6 or 7
bases.
Start codon: AUG codes
for the 1st amino acid,
always a modified
methionine (N-formyl
methionine, fMet).
Codons: Each triplet
of bases following
the start codon codes
for one amino acid.
This codon sets the
reading frame.
S
CH3
S
CH3
Translation
performed by
appropriately
charged tRNAs.
O
H
O
N
H
O
N-formylMet
Ribosome - The ribosomal subunits and their
RNA components are named for their
sedimentation coefficients, S, which is a
measure of how rapidly they move when a
centrifugal force is applied. 50s+30s=70s!
O
H2N
O
Met
50S Subunit


31 Proteins
23S and 5S rRNA
Polypeptide Synthesis:
1. The polypeptide grows stepwise as a
ribosome-bound, peptidyl-tRNA. Discrete
sites on both ribosomal subunits are involved 30 S Subunit
in elongation.
 21 proteins
a) The A site binds the aminoacyl-tRNA
 16S rRNA - binds to Ribosome
(charged tRNA) corresponding to the next
Binding Site (SD) on mRNA.
amino acid.
b) The P site holds the peptidyl-tRNA at
the start of each cycle - the growing chain.
c) The E site is the exit site for the uncharged tRNA, site after peptide
bond formation.
3
Stop codon:
Signals end of the
protein (UAG,
UAA, UGA)
Protein is
hydrolyzed
from last
tRNA.