DNA

TRANSCRIPTION
1 RNA is transcribed
from a DNA template.
3′
A
ly-
Po

RNA RNA
5′
transcript polymerase

RNA PROCESSING
Exon
2 In eukaryotes, the RNA transcript
RNA transcript (pre- (pre-mRNA)
mRNA) is spliced and
modified to produce Intron
mRNA, which moves
from the nucleus to the Aminoacyl-tRNA
cytoplasm. -A synthetase
Poly
NUCLEUS

Amino
CYTOPLASM acid AMINO ACID ACTIVATION
tRNA 4 Each amino acid
3 The mRNA leaves attaches to its proper tRNA

DECIPHERING THE
the nucleus and with the help of a specific
attaches to a ribosome. enzyme and ATP.
mRNA
Growing
polypeptide
p

GENETIC CODE
Ca 3′
5′
A
A
Aminoacyl ly-
P (charged) Po
Ribosomal tRNA
E
subunits

DNA SEQUENCE SPECIFIES PROTEINS

ap
5′ C

TRANSLATION
C U
A C A 5 A succession of tRNAs
C add their amino acids to
E A
Anticodon the polypeptide chain
A A A
as the mRNA is moved
U G G U U U A U G through the ribosome
one codon at a time.
Codon When completed, the
polypeptide is released
from the ribosome.
Ribosome
 TRANSLATION

•  Gene to Protein

•  Decoding – DNA code to Functional Protein

•  DNA Sequence of A,T,G & C to the 20 or so

aminoacids
 INITIAL ATTEPMTS

•  RNA Tie Club – George Gamov

•  Information Storage and
Transfer in Living Cells
•  Recipient of UNESCO Kalinga
Prize
•  20 + 4 members - Watson (PRO)
Crick (TYR)
•  Crick – Adapter
•  Gamov - Triplet
 THE MESSENGER

Lac -

Lac +

Paul Zamecnik Cell Free extract

PaJaMo
Pardee/ Jacob/Monod
 RIBOSOME VS PLANT VIRUS

Both viruses and ribosomes were small, spherical structures, relatively

stable, turning over much less rapidly than the rate of protein synthesis.
RNA was sufficient to determine the nature of the protein synthesized by
the viruses

It was easy to imagine ribosomes as

viruses put in the employ of the cell --
and viruses as ribosomes gone wild.

“...I suspect [ribosomes] have a simple structure (akin to plant viruses),”

he wrote in 1956 to Sydney Brenner.

Francis Crick
 THE MESSENGER

Sucrose Gradient
S35 RNA band + Ribosomes

Wash S35
Soluble fraction

Does the RNA in Ribosome (rRNA) carry the code ?

 DISCOVERY OF MESSENGER

N14 & C14

N15 & C13

Heavy Ribosomes
C14 RNA
C14 RNA
Ribosomes
- Similar to the phage DNA
RNA + Ribosomes

RNA
JACOB/MESELSON/BRENNER
 THE READING FRAME
A to Protein
•  Tripet Non-overlapping Code
ny one of three differ- 5¢ 3¢
rocess begins (Figure CUC AGC GUU ACC AU
1
frames in an mRNA Leu Ser Val Thr
punctuation signal at
g frame at the start of
C UCA GCG UUA CCA U
2
Ser Ala Leu Pro
n mRNA
nize the amino acids CU CAG CGU UAC CAU
xample, bind directly 3
Gln Arg Tyr His
protein depends on
o the codon and, at Figure 6–51 The three possible reading
aptors consist of a set
 THE TRIPLET CODE

Wild-type

CAU CAU CAU CAU CAU CAU

Insertion
CAU ACA UCA UCA UCA UCA U
✔ ✗ ✗ ✗ ✗

Isolation of Revertants
Deletion
CAU ACA UCU CAU CAU CAU
✔ ✗ ✗ ✔ ✔
 THE TRIPLET CODE

Wild-type
CAU CAU CAU CAU CAU

Insertion
CAU ACA CUC TAU CAU CA U
✔ ✗ ✗ ✗ ✔ ✔

Deletion
CAU AUC UCA CAU
✔ ✗ ✗ ✔
 THE TRIPLET CODE

Isolation of Revertants in Lysozyme

Sequence of lysozyme - George Streisinger

 RNA SYNTHESIS

Severo Ochoa

RNA Synthesis

His advice to the science students regarding conductions of research reads as follows :
“ My advice to students of science is that if they have an urge to do research they should to it by all
means. Nothing should stand in the way of a strong wish to devote Life to Science.”
“ If you have the urge to do scientific research get the proper training and by all means do it; nothing
else is likely to give you so much satisfaction and, above all such a sense of fulfilment.”
DECIPHERING THE CODE

POLYNUCLEOTIDE
PHOSPHORLASE
+ Mg2+
nNpp NpNpNp…….n + np
 THE INTRUDER

•  Marshall Nirenberg & Johann H. Matthaei

•  Cell-Free System
•  Strands of template with a known combination of
nucleotides
•  Radiolabled amino acids

The Poly-U experiment

mRNA uuuuuuuuuuuuuuuuuuuuuuuu

Phe-Phe-Phe----- Phenylalanine
DECIPHERING THE CODE

Nucleic Acids to Amino Acids: DNA

Specifies Protein
By: Ann P. Smith, Ph.D. (Write Science
Right) © 2008 Nature Education 

Citation: Smith, A. (2008) Nucleic acids
to amino acids: DNA specifies
protein. Nature Education 1(1):126
 (as 1) (as 100)
(1) 3A 1. AAA 5/6 × 5/6 × 5/6 = 125/216 125 100
(2) 2A1C 2. AAC 5/6 × 5/6 × 1/6 = 25/216 25 20
3. ACA 5/6 × 1/6 × 5/6 = 25/216 25 20

(3) 1A2C
DECIPHERING THE CODE
4. CAA 1/6 × 5/6 × 5/6 = 25/216
5. CCA 1/6 × 1/6 × 5/6 = 5/216
25
5
20
4
6. CAC 1/6 × 5/6 × 1/6 = 5/216 5 4
7. ACC 5/6 × 1/6 × 1/6 = 5/216 5 4
¾ U +(4)¼ G
3C 8. CCC 1/6 × 1/6 × 1/6 = 1/216 1 0.80

CodonThe calculated relative proportions of codons were compared

Probability with the proportions in which
Ratio*
different aminop(UUU)
UUU acids were=3/4 × 3/4 × 3/4 =27/64
present in the polypeptides synthesized 1.00
using poly AC. For instance,
Amino acid ifRatio*
an
amino acid is 1/5th
UUG p(UUG)of lysine (coded by AAA), we can say that it should
=3/4 × 3/4 × 1/4 =9/64 0.33 be coded by one of the three
possible 2A1Cp(UGU)
UGU codons (AAC, ACA or CAA). Similar reasoning would
=3/4 × 1/4 × 3/4 =9/64 Phenylalanine
0.33 allow assignments 1.00
of 1A2C
GUU
as well as 3C. p(GUU) =1/4 × 3/4 × 3/4 =9/64
However, using to assign theLeucine
this technique, it was not possible 0.33 three codons of0.37the
UGG Valine 0.36
category 2A1Cp(UGG)
(i.e., AAC,=3/4 × 1/4 × 1/4 =3/64
ACA, CAA) to three amino acids, since 0.11these will be present in equal
GGU p(GGU)the =1/4 × 1/4 × 3/4 =3/64 Cysteine
0.11respect to 0.35
quantities. Therefore, codons were initially assigned only with base composition,
Tryptophan 0.14
GUG p(GUG) of =1/4 × 3/4 × 1/4 =3/64
ignoring the sequences the bases in codons, as done in the above0.11
example. Glycine
The assignments0.12 are
GGG p(GGG) =1/4 × 1/4 × 1/4 =1/64 0.03
given in Table 30–2.
Table 30–2. Codon assignments derived due to use of A : C (5 : 1) in the synthesis of mRNA

Amino acids Codon composition

(1) lysine 3A
(2) asparagine, glutamine and threonine 2A1C
(3) histidine, proline and threonine 1A2C
(4) proline 3C
B. Assignment of Codons with Known Sequences
1. Binding Technique : Marshall W. Nirenberg and Philip Leder in 1964 found that if a
synthetic trinucleotide for a known sequence (with known bases at 5’ end and 3’ end) is used with
 –A A G/ A A G/ A A G / A A G– or –A/ A G A / A G A / A G A / A– or –A A / G A A / G A A / G A A / G–
d Termination
Translation on
Yields Yields Yields ribosomes in a
cell-free system
–Lys–Lys–Lys–Lys– –Arg–Arg–Arg– –Glu–Glu–Glu–
Polylysine Polyarginine Polyglutamate
urse (a) Fig.UCUCUCUCUCUC
30–8. Use of synthetic polynucleotides with repeating sequences to decipher the code
This example shows how polypeptides derived from the (AAG)n polymer were used to confirm the triplet
re- code andSer Leu codons.
help identify Ser Leu The polymer (AAG)n can yield 3 different polypeptides, depending on
ult, which reading frame is employed.
Using synthetic DNA, Khorana and his coworkers could prepare polyribonucleotides (RNA)
ctu- with known repeating sequences. A repeating sequence means that, if CU are two bases, these will be
do- (b) repeatedlyUUCUUCUUCUUC
present throughout the length as follows :
or
CUCUCUCUCUCUCU UUCUUCUUCUUC
ons. In a similar manner, if ACU are three bases, they will be present repeatedly as follows :
Phe Phe Phe Phe ACUACUACUACU Ser Ser Ser
, so Such copolymers will direct the incorporation of amino acids in a manner which can be theoreti-
cally predicted. For instance, in (CU)n =(CUC/UCU/CUC/UCU), only two codons are possible and
these are CUC and UCU. Moreover, these codons are present in alternating sequence. The result
rich orwould be that
UUCUUCUUCUUC
the polypeptide formed would have only two amino acids in alternating sequences.
These two amino acids can be assigned to the two codons (Table 30–3).
laid Table 30–3 Leu LeuofLeu
Assignment codons, having known sequences, with the help of copolymers
ode having repetitive sequences of two bases.

was Copolymers Codons Amino acids Codons

(CU)n CUC/UCU/CUC leucine/serine CUC/UCU
d be (c) (UG)n
UAUCUAUCUAUC
UGU/GUG/UGU cysteine/valine UGU/GUG
(AC)n ACA/CAC/ACA threonine/histidine ACA/CAC
and Tyr Leu Ser Ile
osed We may similarly consider a repeating sequence of three bases e.g., (ACG)n. Depending upon
Figurewhere
18.4the Coding
reading is started, three kinds
properties 45 ofofseveral
homopolypeptides
deciphered
are expected
synthetic mRNAs. (Table 30–4). Actual
that codon assignment i.e,. to find out which of the three codons codes for which amino acid would
(a) Poly(UC) contains twoinformation
alternating codons, UCU and CUC, which
depend upon the previous available regarding the composition of bases in different
U’s. code for serine
codons coding(Ser) and amino
for different leucineacids.(Leu), respectively. Thus, the product
 2. Repetitive Sequencing Technique: This degree from Punjab
method of confirming the genetic code is the most direct University and a Ph.D.
method and was devised by the Nobel prize winner Prof. from the University of
Har Gobind Khorana. This method consists of in vitro Liverpool. In 1960 he
chemical synthesis of short segments of DNA of known joined the faculty at the
DECIPHERING THE CODE
base sequence with the help of DNA polymerase. From University of Wisconsin
this synthetic DNA, a polyribonucleotide (RNA) of and later became a
strictly defined base sequence is transcribed under the professor at MIT.
catalytic influence of RNA polymerase. A polypeptide
is then synthesised under the direction of RNA as represented in Fig. 30–7.
Short polydeoxyribonucleotide
of known base sequence
Contents
RNA polymerase

DNA polymerase Long polyribonucleotide in vitro Polypeptide of

of known sequence protein synthe- known sequence
822 FUNDAMENTALS OF BIOCHEMISTRYRNA polymerase
sizing system
Long polydeoxyribonucleotide
did not revealbase
of known which codon corresponded to which amino acid; further experiments were needed to
sequence
discriminate amongFig.the 30–7.
possible matches.
Synthesis of polypeptide under the direction of RNA
The synthetic polynu- can be read in three
The amino acid sequence
cleotide (AAG)n of the polypeptide so formed
–A A G A A G A A G A A G– is then
different determined and correlated with
frames
the base sequence of DNA and RNA.
Using homopolynucleotides (all bases the same) as templates could yield only a few code words.
Many more–A A G/words
A A G/ Awere
A G / Adeciphered after
A G– or –A/ A G A/AG Khorana developed
A / A G A / A– or –A A / G Amethods
A / G A A / for
G A Asynthesizing
/ G–
polyribonucleotides with different but repeating structures. In the example shown in Fig. 30–8, the
Translation on
(AAG)n was found to give
repeating sequenceYields 3 different homopolymersYields
Yields : polylysine,
ribosomes polyarginine
in a
and polyglutamic acid. This finding not only confirmed the importance of the reading frame but also
cell-free system
showed that AAG, AGA and GAA must be
–Lys–Lys–Lys–Lys– codons
–Arg– Arg–Argfor
– these amino acids. However, the experiment
–Glu–Glu–Glu–
Polylysine Polyarginine Polyglutamate
Fig. 30–8. Use of synthetic polynucleotides with repeating sequences to decipher the code
This example shows how polypeptides derived from the (AAG)n polymer were used to confirm the triplet
code and help identify codons. The polymer (AAG)n can yield 3 different polypeptides, depending on
 (CU)n CUC/UCU/CUC leucine/serine CUC/UCU
(UG)n UGU/GUG/UGU cysteine/valine UGU/GUG
(AC)n ACA/CAC/ACA threonine/histidine ACA/CAC

We may similarly consider a repeating sequence of three bases e.g., (ACG)n. Depending upon
where the reading is started, three kinds of homopolypeptides are expected (Table 30–4). Actual
codon assignment i.e,. to find out which of the three codons codes for which amino acid would
depend upon the previous information available regarding the composition of bases in different
codons coding for different amino acids.
Table 30–4. Assignment of codons, having known sequences, with the help or
copolymers having repetitive sequeuces of three bases = (ACG)n.
Codons Homopolypeptide Codon assignment
ACG/ACG/ACG/ACG/ACG = Poly (ACG) (threonine)n ACG = threonine
A/CGA/CGA/CGA/CGA/CGA = Poly (CGA) (arginine)n CGA = arginine
AC/GAC/GAC/GAC/GAC = poly (GAC) (aspartic acid)n GAC = aspartic acid

These studies of Khorana and his coworkers with chemically-defined messengers proved very
conclusively that (1) the base sequence in DNA specifies the sequence of amino acids in proteins, (2)
 AN ADAPTER TO READ THE CODE
abotism

ER lumen
Amino acid

Amino acid
binding site

mRNA
Xrr.l"otid" trlpt"t
coding for an
amino acid

FIGURE 27-2 Crick's adaptor hypothesis.Todaywe know that the

d endoplasmic reticulum.Electronmicro-
ngof a portionof a pancreatic
cell,show-
eouter(cytosolic)faceof theendoplasmic
aminoacid is covalentlyboundat the 3' end of a IRNAmoleculeand
thata specificnucleotide
tripletelsewherein theIRNAinteracts
with a
 ROLE OF THE ADAPTER

Discovery of Aminoacyl tRNA Synthetases & tRNA (sRNA)

Paul Zamecnik , Mahlon Hoagland & Mary Stephensen

 ROLE OF THE ADAPTER

Nickel Hydride
CYSTEINE + tRNACys ALANINE - tRNACys

UGU UGU

CYS ALA
 RIBOTRINUCLEOTIDES

RNase A
ApG + U 2’ 3’ UpApG + (Up)nApG
CYCLIC PHOSPHATE

POLYNUCLEOTIDE
PHOSPHORLASE
+ Mg2+
ApG + NDP ApGpN + ApG(pN)n + p

XpY + NDP XpYpN + XpY(pN)n + p

RIBO-TRINUCLEOTIDES

4 x 4 x 4 = 64
All 64 TRIPELTS
 DECIPHERING THE CODE

Nucleic Acids to Amino Acids: DNA Specifies Protein

By: Ann P. Smith, Ph.D. (Write Science Right) © 2008 Nature
Education 

Citation: Smith, A. (2008) Nucleic acids to amino acids: DNA

specifies protein. Nature Education 1(1):126
 THE RIDDLE IS SOLVED

Robert William Holley Har Gobind Khorana Marshall Warren Nirenberg

1922 - 1993 1922 - 2011 1927 - 2010
the mRNA,all the subsequentcodonswill be out of reg- the absenceof initiationcodons.
ister; the result is usually a "missense"protein with a imental conditionscausedthe no
garbledaminoacid sequence. ments for protein synthesisto
Severalcodonsseruespecialfunctions(trig.27-T). combinedweth chanceto produ
The initiation codon AUG is the most commonsignal cornmonoccurrenceur the histor
THE CODE
for the beginningof a polypeptidein all cells,in addition
to codrng for Met residues in internal positions of
In a randomsequenceof nuc
codonsin eachreadingframe is,
tion codon.In general,a reading
nation codonamong50 or more c
First letter ofcodon(5'end)
I an open reading frarne (ORF
I Secondletter frames usually correspondto g
I ofcodon
l€ teins. In the analysisof sequen
YUCA
cated programsare used to se
UUU Phe UCU Ser UAU Tyr UGU cy. frames in order to find genes a
UUC Phe UCC Ser UAC Tyt U G C cy.
U
background of nongenic DNA. A
UUA Leu UCA Ser UAA Stop UGA Stop coding for a typical protein with
UUG Leu UCG Ser UAG Stop UGG T.p 60,000would requirean openrea
CUU Leu CCU Pro CAU His CGU Arg morecodons.
CUC Leu CCC Pro CAC His CGC Arg A striking feature of the g
aminoacidmay be specifledby m
CUA Leu ccA Pro CAA Gln CGA Arg
CUG Leu ccG Pro CAG Gln CGG Arg the codeis describedasdegener
gest that the codeis flawed:altho
AUU Ile ACU Thr AAU Asn AGU Ser havetwo or more codons,eachc
AUC Ile ACC Thr AAC Asn AGC Ser
aminoacid.The degeneracyof th
AUA Ile ACA Thr AAA Lys AGA Arg Whereas methionine and try
AUG Met ACG Thr AAG Lys AGG Arg codons,for example,three amin
GUU Val GCU AIa GAU Asp GGU Gly have six codons,flve amino acid
GUC Val GCC Ala GAC A.p GGC Gly has three, and nine aminoacids
The genetic code is nearly
GUA Val GCA Ala GAA Glu GGA Gly
GUG Val GCG AIa GAG Glu GGG GIY triguing exceptionof a few minor
dria, somebacteria,and some s
FlfrURl?7-i "Dictionary"of aminoacidcodewordsin mRNAs.The
(Box 27-l), aminoacidcodonsa
codonsare written in the 5'-+3'direction The third baseof each examinedso far. Humanbeings
and virusessharet
 THE EMPTY CODONS

UAG
Ochre
UAA

UGA

Richard Epstein and Charles Steinberg

Harris Bernstein
Immer Wieder Bernstein – “Forever Amber” Sydney Brenner
 THE STOP SIGNAL

m0 su+

m1 su+

m2 su+

m3 su+

m4 su+

m5 su+ UAG & UAA

m6 su+

m su-
S W Y E L Q
324_ch18_560-600.indd Page 568 12/16/10 10:54 AM user-f469 /Volume/204/MHDQ268/wea25324_disk1of1/00735253

568 EXCEPTIONS TO THE CODE

Chapter 18 / The Mechanism of Translation II: Elongation and Termination

Table 18.1 Deviations from the “Universal” Genetic Code

Source Codon Usual meaning New meaning

Fruit fly mitochondria UGA Stop Tryptophan

AGA & AGG Arginine Serine
AUA Isoleucine Methionine
Mammalian mitochondria AGA & AGG Arginine Stop
AUA Isoleucine Methionine
UGA Stop Tryptophan
Yeast mitochondria CUN* Leucine Threonine
AUA Isoleucine Methionine
UGA Stop Tryptophan
Higher plant mitochondria UGA Stop Tryptophan
CGG Arginine Tryptophan
Candida albicans nuclei CTG Leucine Serine
Protozoa nuclei UAA & UAG Stop Glutamine
Mycoplasma UGA Stop Tryptophan

*N 5 Any base.

What* about
21st &
the22 nd aa -that
argument Selenocysteine
the code is random:&that
Pyrrolysine
25 (Coded by STOP Codons)
the existing codons have no inherent advantage? Actually,
when we consider the code’s effectiveness in dealing with
EXCEPTION OR NORM !!!
RECODING