Nobel prize (1968) to
Nirenberg & Khorana
The Genetic Code
• Nonoverlapping: 1 codon follow another
• Triplet of nucleotides = one codon
• Degenerate: more than one codon may direct more than
one amino acid
• Linear: A linear piece of DNA contains the genetic
information which is transferred to the RNA transcript
which then directs ptn synthesis.
• Code is unambiguous: all 64 possible codons are
defined (either an amino acid or a stop codon)
•Initiation (1 start codon) and termination (3 stop codons)
• ‘Commaless’ Code read from start to end. No stopping
or jumping about (introns already removed).
•Universal: All organisms use same basic genetic code
(but some minor exceptions).
Overlapping vs. Nonoverlapping
Nonoverlapping = 1 base mutation = 1 amino acid changed.
Overlapping = 1 base mutation = as many as 3 amino acids changed.
Evidence for Triplet Codons use
Mathematical considerations: There are at least 20
amino acids that have to be encoded
1 nt code = 4 amino acids possible
2 nt code = 16 amino acids possible
3 nt code = 64 amino acids possible
Thus, 3 is enough to code for all 20 amino acids.
Suppressor mutations with T4 rII locus: early evidence for a triplet code
Isolated revertants: These were rII+ due to a 2nd mutation…they could
separate this out by phage crosses with a true wt rII+
FCO-> a proflavin induced mutation (thought to cause deletions and insertions of single bases).
NOT the same as WT!
The revertant LOOKED like wt phenotypically but when Crick et al crossed these with
WT rII phage, they got back two independent mutants. These could be mapped again
by Benzer’s crossing system….it appears that the gene is ‘polarized’.
MODEL: Suggested that the ORF was ‘frameshifted’ with inserting or deleting a base….
•Insert or delete 1 nucleotide ==> nonfunctional
•Get revertant by doing the opposite (delete or insert 1 nucleotide..restores ORF).
•Insert or delete 2 nucleotides ==> nonfunctional (not doublet codon)
•Insert or delete 3 nucleotides ==> partly functional, restores the reading frame.
This was strong evidence for a triplet codon.
•Deletion = -1
•Insertion = +1
•Add deletions and insertions!
•If = 0, (+/-)3, (+/-)6, etc. then reading frame restored.
What was established
• Code is nonoverlapping
• Three bases = one amino acid
• MW of 3 bases is about 1000, MW of 1 amino acid is
• Code is read from a fixed point and read to the end of
the mRNA since a single base deletion or addition alters
the downstream alignment of the coding sequence
• The code is degenerate: 43 possibilities = 64 drives 20
aa. Some specificied by the same codon
Cracking the Code:
Synthesis of RNA polymers
Polynucleotide phosphorylase: Template independent RNA synthesis
-This reaction is more favorable in the degradation direction (with large excess of
ribonucleotide diphosphates can force the arrow in the synthesis direction.)
-Reaction carried out in a cell-free system to make short, synthetic mRNA.
-These mRNA’s can be used to make proteins.
- Ribonucleotides are added in a random fashion, based on the relative
concentration of the 4 ribonucleotide diphosphates in the reaction mixtures.
- Add the synthetic RNA to cell extract (E. coli): direct the synthesis of proteins.
- This is an in vitro (cell-free) system.
Cracking the Code: Mixed RNA copolymers
RNA with fixed
base ratios for ex:
1/6 A 5/6 C
His: 14% = 11.6 + 2.3
Table 10.3 & 10.4
Proline: 69% = 57.9
tRNA Recognition of the codons
• Used clever chemistry to convert a bound
amino acid on a charged tRNA:
In vitro TLN would incorporate ala, not cys.
Shows that amino acids are unable to insert alone and
require an adaptor molecule of tRNA.
The tRNA recognizes and binds a specific aa
The anticodon in tRNA binds the mRNA to align the aa
with the growing polypeptide chain during TLN
1. Anticodon: C’ to codon and antiparallel
2. Amino acid attachment site
3. Loops (arms) aid in ribosome alignment
Encoded by nuclear genes: example of a
‘gene product’ that is not protein
tRNA folding is crucial and not random
Genetic Code Table
1st base of 3rd base of
anticodon codon (3’ end)
U A or G
G U or C
I U, C, or A
I = inosine
Also see Fig. 10.28,
Table 10.5, & Table 10.6
Multiple codon use: Degeneracy of the genetic code
-Number of codons for each aa varies from one to 6
-Certain aa can be delivered to the ribosome by several alternative tRNA
species having different anticodons
-Certain tRNA species can bring their SPECIFIC amino acids in response to
several DIFFERENT codons (not just one). This is due to ‘slop’ in the
pairing of the 3rd base also called ‘wobble’.
THERE ARE SOME RULES THAT DICTATE THE WOBBLE POSITION
In the 5’ anticodon, a G can
hybridize with U or C
-This anticodon can therefore
recognize either UCU or UCC
Different tRNA species may accept or recognize the
same amino acid:
These tRNA are encoded by different nuclear genes.
Isoaccepting tRNA species
• Some codons STOP or terminate TLN
• Identified using Benzer’s T4 system with a
phage head ptn called m
• m mutants gave truncated proteins
• Data suggested that UAG (amber) was a stop
(based on analysis of aa at the termination
point, see Fig. 10-29)
• Others are opal (UGA) and ochre (UAA)
• Such non-sense codons can be suppressed by
secondary mutations elsewhere.
DNA 3’ TACGCTAGCATC 5’ template strand
Same as the
RNA 5’ AUGCGAUCGUAG 3’ nontemplate strand
except for “U” in the
place of “T”.
Protein N- met-arg-ser -C
Components of Translation
•mRNA (with many triplet codons): Assembly
point for all below.
•Ribosomes: Machinery where TLN occurs
•tRNA: “adaptor” molecules
•Aminoacyl tRNA synthases: Catalyze the attachment (‘charge’
the tRNA) of each amino acid to its corresponding tRNA molecule.
aa1 + tRNA1 = ATP synthetase1 aa1-tRNA1 + AMP +PPi
• Initiation factors
•Elongation factors Protein Synthesis: Chemical reaction that makes
peptide bonds between aa residues
OVERVIEW of PROCESS
1. Charge the tRNA with an aa residue using a specific tRNA synthetase
aa1 + tRNA1 = ATP synthetase1 aa1-tRNA1 + AMP +PPi
2, Energy of charged tRNA used to make a peptide bond: Uses peptidyl
transferase on ribosome
3. New residues added in succession
Ribosomes in Prokaryotes and Eukaryotes: a comparison
•The assembly of ribosomes onto RNA transcripts can be seen in an electron
•Ribosomes have 2 main subunits: large and small, each made up of a combination
of proteins and rRNA’s.
•The various subunits are named according to their behavior in a sucrose density
gradient. The faster it travels (rate of sedimentation) the larger the S (Svedberg)
•The E. Coli genome contains 7 copies of a single gene sequence containing all 3
rRNA genes. This sequence is transcribed as a unit and processed into the 3 rRNAs
(23S, 16S, and 5S).
Ribosomes in Prokaryotes and Eukaryotes: a comparison
•large subunit = 49 proteins + rRNA (28S + 5.8S + 5S)
•small subunit + 33 proteins + 18S rRNA
•In eukaryotes there are greater than 100 copies of the rRNA genes.
Again, a single transcript is transcribed and processed into three rRNAs
(28S, 18S, and 5.8S). The 5S rRNA is transcribed from a separate gene.
3 parts to translation: initiation, elongation, and termination.
Mostly focus on translation in prokaryotes,
AUG = start codon. It tells the ribosome where to start translation.
Translation does not start at the very 5’ end of the mRNA.
The region upstream of the AUG start codon is called the 5’ untranslated region.
The ribosome scans the mRNA for an AUG in the correct context.
- AUG codes for met and met can also be found in the middle of a protein.
How does the ribosome know which AUG to use?
There is a sequence (called Shine-Dalgarno sequence in prokaryotes, AGGAGG) that
immediately precedes the start AUG (see Fig. 10-34).
This sequence complimentary binds to the 16S rRNA found in the small ribosomal
subunit to initiate translation
at the correct AUG.
SD start stop
5’untranslated region coding region 3’untranslated region
• In prokaryotes formylmethionine (f-met) is the first amino acid added. A
special tRNA fmet is used as the first charged tRNA (initiator tRNA).
• Eukaryotes use the Kozak sequence to define the correct AUG to start at. If
the Kozak sequence is mutated then the AUG cannot be used as the start
codon. ACCAUGG = Kozak.
• The ribosome has 3 binding sites for tRNA:E = exit P = peptidyl A =
Step 1: Initiation
IF1, IF3 = bind small ribosomal
IF2 = binds tRNAfmet
are ‘free’ or
future P site
Step 1: 30s binds to mRNA TLN on mRNA
stimulated by IF3
Step 2: IF2/GTP + fMet-tRNA leads
to occupation of fMet-tRNA at P
site (P = peptidyl)
Step 3: Hydrolysis of GTP drives
assembly of 50s ribosomal unit to
give the 70s
Step 2: Elongation
1. EF-Tu mediates entry of
charged tRNAs into A site
(requires GTP co-factor)
2. EF-Ts helps regenerated EF-
transfers nascent chain from
P to A site (c). EF-G
mediates translocation and
release of tRNA
New (nascent) chain now at P
site ready to go again.
• Note that the mRNA is read in the 5’
to 3’ direction and the protein grows
in the N-terminal to C-terminal
• Note that throughout translation the
energy comes from GTP. The usual
energy currency of the cell is ATP.
Translation (Step 3): Termination Fig. 10.37
Elongation will continue until a STOP codon (UAA,
UAG, or UGA) is encountered.
1. When a STOP codon is reached there is no
tRNA to add to the “A site”. Rather a release
factor (RF1, RF2, RF3) can interact with the “A
site” and fill it. There is no amino acid on the
termination factor to add to the peptide chain, thus
the newly synthesized protein is released with the
help of release factors. This requires energy from
2. After the release of the protein, the last
uncharged tRNA is released and the ribosome falls
apart releasing the large and small subunits. The STOP Codons
release factor is also released. All of these factors UAA
can be reused in the synthesis of other proteins. UAG
Note that there are sequences downstream of the UGA
termination codon. This is called the 3’
Mediated by release factors.
Energy from GTP.
Peptide bond formation overview
http://www.biosci.ohio-state.edu/mg606 Go to Lecture 7: translation
Non-sense suppressor mutation: Brenner et al. found
suppressors that could give WT phenotypes.
Below: TLN stops at UAG since there is tRNA that recognizes
UAG ‘amber’ codon = termination and release
Non-sense suppressor mutation: by mutation a tRNA itself, the
cell now has a ‘new’ tRNA that can bypass a non-sense
mutation…we say it’s an amber suppressor.
Here is how it may work for a Tyrosine tRNA suppressor:
Suppressor in action: This mRNA has a UAG that should
halt TLN…but instead, a TYR is inserted in the chain!
• The new polypeptide may be further
modified by addition of leader peptide or
signal sequence (25 aa long, hydrophobic in
nature for insertion into membranes)
• Some Proteins made as larger ‘pro-forms’
that are proteolytically cleaved or matured.
• Intervening ptn sequences: spliced out to
form a processed product (usually
Read over ‘universality’ of the
genetic code (Fig. 10-43)
• TLN is generic and uniform across many
Functional division of labor in the
• Era of genomics: fundamental questions such
as # of genes required to define an organism
• Entire protein coding potential of a genome is
collectively called a ‘proteome”
• Yeast: over 6000 genes
• Worm: (C. elegans): >19K genes
• A breakdown of metabolism and proteome is
given in Fig. 10-44 and 45.