Chapter 22 (Part 1)
Protein Synthesis
Translating the Message
• How does the sequence of mRNA translate into the
sequence of a protein?
• What is the genetic code?
• How do you translate the "four-letter code" of
mRNA into the "20-letter code" of proteins?
• And what are the mechanics like? There is no
obvious chemical affinity between the purine and
pyrimidine bases and the amino acids that make
protein.
• As a "way out" of this dilemma, Crick proposed
"adapter molecules" - they are tRNAs!
The Collinearity of Gene
and Protein Structures
• Watson and Crick's structure for DNA,
together with Sanger's demonstration that
protein sequences were unique and specific,
made it seem likely that DNA sequence
specified protein sequence
• Yanofsky provided better evidence in 1964: he
showed that the relative distances between
mutations in DNA were proportional to the
distances between amino acid substitutions in
E. coli tryptophan synthase
Elucidating the Genetic Code
• How does DNA code for 20 different
amino acids?
• 2 letter code would allow for only 16
possible combinations.
• 4 letter code would allow for 256
possible combinations.
• 3 letter code would allow for 64
different combinations
• Is the code overlapping?
• Is the code punctuated?
The Nature of the Genetic Code
• A group of three bases codes for
one amino acid
• The code is not overlapping
• The base sequence is read from a
fixed starting point, with no
punctuation
• The code is degenerate (in most
cases, each amino acid can be
designated by any of several
triplets)
How the code was broken
• Assignment of "codons" to their respective
amino acids was achieved by in vitro
biochemistry
• Marshall Nirenberg and Heinrich Matthaei
showed that poly-U produced
polyphenylalanine in a cell-free solution from
E. coli
• Poly-A gave polylysine
• Poly-C gave polyproline
• Poly-G gave polyglycine
• But what of others?
Getting at the Rest of the Code
• Work with nucleotide copolymers (poly (A,C),
etc.), revealed some of the codes
• But Marshall Nirenberg and Philip Leder
cracked the entire code in 1964
• They showed that trinucleotides bound to
ribosomes could direct the binding of specific
aminoacyl-tRNAs
• By using C-14 labelled amino acids with all
the possible trinucleotide codes, they
elucidated all 64 correspondences in the code
Features of the Genetic Code
• All the codons have meaning: 61 specify amino
acids, and the other 3 are "nonsense" or "stop"
codons
• The code is unambiguous - only one amino acid is
indicated by each of the 61 codons
• The code is degenerate - except for Trp and Met,
each amino acid is coded by two or more codons
• First 2 codons of triplet are often enough to specify
amino acid. Third position differs
• Codons representing the same or similar amino acids
are similar in sequence (Glu and Asp)
tRNAs
• tRNAs are interpreters of
the genetic code
• Length = 73 – 95 bases
• Have extensive 2o
structure
• Acceptor arm – position
where amino acid
attached
• Anticodon –
complementary to mRNA
• Several covalently
modified bases
• Gray bases are conserved
between tRNAs
tRNAs: 2o vs 3o Structure
Third-Base Degeneracy
• Codon-anticodon pairing is the crucial
feature of the "reading of the code"
• But what accounts for "degeneracy": are
there 61 different anticodons, or can you
get by with fewer than 61, due to lack of
specificity at the third position?
• Crick's Wobble Hypothesis argues for the
second possibility - the first base of the
anticodon (which matches the 3rd base of
the codon) is referred to as the "wobble
position"
The Wobble Hypothesis
• The first two bases of the codon make normal
H-bond pairs with the 2nd and 3rd bases of
the anticodon
• At the remaining position, less stringent rules
apply and non-canonical pairing may occur
• The rules: first base U can recognize A or G,
first base G can recognize U or C, and first
base I can recognize U, C or A (I comes from
deamination of A)
• Advantage of wobble: dissociation of tRNA
from mRNA is faster and protein synthesis
too
AA Activation for Prot. Synth.
• Codons are recognized by aminoacyl-tRNAs
• Base pairing must allow the tRNA to bring its
particular amino acid to the ribosome
• But aminoacyl-tRNAs do something else: activate
the amino acid for transfer to peptide
• Aminoacyl-tRNA synthetases do the critical job
- linking the right amino acid with "cognate"
tRNA
• Two levels of specificity - one in forming the
aminoacyl adenylate and one in linking to tRNA
Aminoacyl-tRNA Synthetase
Amino acid + tRNA + ATP  aminoacyl-tRNA + AMP + PPi
•
Most species have at least 20 different aminoacyltRNA synthetases.
•
Typically one enzyme is able to recognize multiple
anticodons coding for a single amino acids (I.e serine 6
different anticodons and only one synthetase)
•
Two step process:
1) Activation of amino acid to aminoacyladenylate
2) Formation of amino-acyl-tRNA
Aminoacyladenylate Formation
NH2
N
N
N
N
O
O
O
H
H OH
H
P
O
O
O-
P
O-
O
O
P
O-
ONH2
H
OH
O
N
N
O
C
CH
N
H
N
O
PPi
NH2
O
O
H
H OH
H
P
O-
O
H
OH
O
C
CH
NH2
H
Aminoacyl-tRNA Synthetase Rxn
NH2
NH2
N
N
N
N
5' tRNA
N
N
O
O
O
H
H OH
H
H
OH
P
O-
O
H
C
N
O
O
O
N
H
H
O
H
OH
H
CH H
NH2
NH3+
N
AMP
5' tRNA
N
O
O
H
O
H
H
O
H
OH
C
CH H
NH3+
N
N
Specificity of AminoacyltRNA Synthetases
• Anticodon and structure features of
acceptor arm of specific tRNAs are
important in enzyme recognition
• Synthetases are highly specific for
substrates, but Ile-tRNA synthetase has
1% error rate. Sometimes incorporates Val.
• Ile-tRNA has proof reading function. Has
deacylase activity that "edits" and
hydrolyzes misacylated aminoacyl-tRNAs