A question always asked..

Why would the cell want to have an intermediate between DNA and the
proteins it encodes?

➢ In Eukaryotes the DNA can stay pristine and protected, away from the
caustic chemistry of the cytoplasm.

➢ Gene information can be amplified by having many copies of an RNA made

from one copy of DNA

➢ Regulation of gene expression can be effected by having specific controls at

each element of the pathway between DNA and proteins. The more elements
there are the pathway, the more opportunities there are to control it in
different circumstances.
The Genetic Code.........

➢ DNA is constructed from nucleotides containing only four possible bases (A. G. C. and
T).

➢ The big question was: how do you code for all of the traits of an organism using only a
four-letter alphabet?

➢ The information stored in DNA is ultimately transferred to protein. which is what gives
cells and tissues their particular properties

➢ Proteins are linear chains of amino acids. and there are 20 amino acids found in proteins.

➢ So the real question becomes: Low does a four-letter alphabet code for all possible
combinations of 20 amino acids?

➢ Marshall Nirenberg shared the 1968 Nobel Prize in Physiology and Medicine with H.
Gobind Khorana for solving the Genetic Code
The Genetic Code……..

➢ By constructing multi-letter “words” out of the four letters in the alphabet, it

is possible to code for all of the amino acids.

➢ Specifically, it is possible to make 64 different three letter words from just

the four letters of the genetic alphabet, which covers the 20 amino acids
easily.

➢ This kind of reasoning led to the proposal of a triplet genetic code.

➢ The Genetic Code......

➢ During translation, information must be translated

➢ From a nucleotide sequence made from 4 different nucleotides

➢ A. C. G. & U to an amino acid sequence made from 20 different amino acids

➢ We have 4 letters to express 20 thoughts

➢ How might the problem be solved?

➢ The language of amino acids is based on codons

1 codon 3 mRNA nucleotide

1 codon 1 Amino acid
The Genetic Code.....

➢ Important elements of the genetic code:

1. The code is a triplet code: Each mRNA codon (word) that specifies a particular
amino acid in a polypeptide chain consists of three nucleotides (letters).

➢ For example, AAG = lysine

2. The code is non-overlapping: The mRNA encoding one protein is read in

successive groups of three nucleotides.

3. The code is degenerate. More an one mRNA codon (word) occurs for the same
amino acids
(ie. AAG and AAA are read both as lysine).
The Genetic Code.

Important elements of the genetic code:

Codon-Anticodon Pairings Allowed by the Wobble Rules

5′ end of anticodon 3′ end of codon

G U or C
C G only
A U only
U A or G
I (Inosine) U, C, or A
The Genetic Code…

➢ Important elements of the genetic code:

➢ a) Wobble - certain different codons are recognized by the same tRNAs

➢ because the 3rd base in the codon and the Ist base of the anticodon pair via a "loose pairing”

➢ This "loose pairing is according to a set of rules known as the wobble rules
The Genetic Code...

➢ Important elements of the genetics code:

➢ b) There are more than one RNA type (therefore more than one anticodon) for some amino acids.

➢ Total 64 codons: Three = stop so total 61 codons, which require 6) tRNA.

➢ But usually there are less than 45 tRNAs in the organisms

➢ 4 The code has start signals (AUG and rarely GUG) and stop signals (UAA. UAG, and UGA Stop
Signals are also called nonsense codons because they do not designate an amino acid

➢ 5 The code is Commaless

➢ 6 The code is almost universal

The Genetic Code...
The Genetic Code....

➢ Nearly all organisms use the same code

➢ Multiple codes can code for the same amino acids. (The code is degenerate)

➢ Variation often occurs in the 3rd letter. Similar codes code for amino acids with similar
physical and chemical properties.

➢ The message is read in non-overlapping codes.

➢ This allows for three possible reading frames.
 The Transfer RNA (tRNA)

➢ The function of tRNA is to ensure that each amino acid incorporated into a
protein corresponds to a particular Codon on the messenger RNA.

➢ tRNA does this by having an anti-codon in one of its loops, which binds to the
codon on the mRNA, and an amino acid at the 3' end of the tRNA
 The Transfer RNA (tRNA)

➢ The correct amino acid is attached to the correct tRNA by an enzyme called
aminoacyl-tRNA synthetase.

➢ Amino acids are put onto the 3'-ends of tRNA molecules (a process called
"charging") by these specific enzymes using ATP energy
 Charging of tRNA:

➢ Transfer RNAS must exist for 20 amino acids.

➢ Sometimes the IRNA anticodon is recognized by a specific enzyme associated with

the proper amino acids.

➢ Sometimes other portions of the RNA act to guarantee interaction

➢ only with the proper enzyme.
 Charging of RNA:

➢ Aminoacyl-tRNA synthetases first associate with proper amino acids and

ATP.

➢ Amino acid is then associated with AMP (ATP loses two phosphates)
forming aminoacyl-AMP.

➢ Next uncharged tRNA associates with enzyme such that 3' end is adjacent
to amino acid.
 Charging of tRNA:

➢ Synthetase then switches aminoacyl bond from AMP to tRNA So

ERNA and amino acid to form proper aminoacyl-tRNA (or aa-tRNA).

➢ Both aa-tRNA and AMP are then released from synthetase.

 The Transfer RNA (tRNA)

➢ There are 20 different aminoacyl-tRNA synthetases, one for each amino acid.

➢ Despite the fact that they all carry out very similar tasks, they vary greatly in
size (40-100 kDalton).

➢ Since there are 61 amino acid codons, most tRNA synthetases must be able to
recognize more than one type of tRNA (i.e. 6 codons for Arg).

➢ These tRNAs are called cognate tRNAs for that particular synthetase
 Primary & Secondary structure of tRNAs:

➢ All tRNAs have similar sequences of about 73 to 93

nucleotides

➢ 3' end always terminates with the sequence CCA, with the 3'
hydroxyl of the ribose of the terminal A being the point of
covalent attachment of the amino acid
 Primary & Secondary structure of tRNAs:

➢ tRNA contains a number (7-15%) of unique/modified bases.

➢ These are post-transcriptionally modified after synthesis by

RNA polymerase.

➢ In particular, adenosine (A) in the first or 5’ position of the

anticodon (corresponding to the third or 3' position of the
codon) is always modified to inosine (I), which lacks the
amino group on the purine ring
 Primary & secondary structure of tRNAs:

➢ Inosine can base-pair with A. U or C and thus

accounts for much of the degeneracy of the
Genetic Code ("Wobble Theory").
 Primary & secondary structure of tRNAs:

➢ tRNAs have cloverleaf secondary structure due to four

base-paired stems

➢ The cloverleaf contains three non-base- paired loops: D.

anticodon. and TΨC loop.
 Primary & secondary structure of tRNAs:

➢ In the cloverleaf model. the inverted sequences form four

double-helical segments.

➢ The two ends of the molecule for the amino acid arm or
acceptor stem.

➢ A single strand sequence - CCA – protrudes from the 3’-end

of the molecule.

➢ The terminal A is coupled to the amino acid during amino

acid activation.
 Primary & secondary structure of tRNAs:

1. The next arm. moving clockwise. is the TΨC arm.

2. The T-loop is also known as the TΨC arm due to the presence of
thymidine. pseudouridine and cytidine residues.

3. This arm got its name from the occurrence of the sequence TPsiC
in almost all tRNAs (Psi. pseudouridine. is a modified U).

4. The next arm is the variable arm which may contain between 4
and 21 nucleotides.
 Primary & secondary structure of tRNAs:

➢ Adjacent to the variable arm is the anticodon arm with the

three-nucleotide anticodon, which pairs with the codon during
translation.

➢ The last arm of the tRNA is the DHU-arm named after the
occurrence of dihydrouridine in all tRNAs.
 A brief overview of the Ribosome

➢ The ribosome is a large and complex molecular machine.

found within all living cells. that serves as the primary site of
biological protein synthesis.

➢ Ribosomes link amino acids together in the order specified

by messenger RNA (mRNA) molecules.
 A brief overview of the Ribosome

➢ Ribosomes consist of two major subunits

➢ The small ribosomal subunit reads the mRNA. while the

large subunit joins amino acids to form a polypeptide chain.

➢ Each subunit is composed of one or more ribosomal RNA

(RNA) molecules and a variety of proteins.

➢ The term originates from ribonucleic acid an the Greek

soma meaning. "body".
Components of the ribosome:

➢ The bacterial (70S) ribosome consists of a small (30S) and a

large (5OS) subunit. With molecular weights of about 800
000 and 1 500 000 Dalton (Da). respectively. where S stands
for the Svedberg unit for sedimentation velocity.

➢ The 30S subunit consists of about 20 different proteins and a

sequence. 16S. of ribosomal RNA (TRNA) containing about
1600 nucleotides.
Components of the ribosome:

➢ The 50S subunit consists of about 33 different proteins, a

23S RNA sequence with about 2900 nucleotides. and a 5S
rRNA sequence with about 120 nucleotides.

➢ Ribosomes from eukaryotes are larger and more complex

than those from prokaryotes. but the ribosomes from all three
kingdoms of life function according to the very same
principles.
Components of the ribosome:

➢ The ribosome has three binding sites for tRNA.

➢ The A (aminoacyl) site

➢ The P (peptidyl) site and

➢ The E (exit) site, formed in the inter subunit interface.

➢ The mRNA binds in a track around the neck of the 30S

subunit, through which it can move in a stepwise manner,
one codon at the time, during peptide elongation of a nascent
chain.
Ribosomes:

➢ Ribosomes occur free in the cytosol or attached to the

endoplasmic reticulum.

➢ rRNAs form the core of ribosomes and are stabilized by

extended base-stacking & base-pairing.

➢ Most ribosomal proteins are located on the surface

➢ Peptide-bond formation is catalysed by a specific adenine

base of the 23S tRNA (ribozyme) in the large subunit.
Prokaryotic vs Eukaryotic Ribosomes
 Crystallographic Pictures of 50S Ribosomes

The path to the SOS subunit structure at high resolution.

The 50S subunit structure at 9A resolution (left, 1998), SÀ resolution (middle, 1999)
and 2.4A resolution (right, 2000) (From Ban et al. 1998; 1999; 2000).
 Translation

➢ Translation is the production of a polypeptide (protein) using RNA as a

template and tRNA molecules as “adapters” that convert the nucleic
acid code to protein code

➢ Factors required for translation:

A. Prokaryotic

1. mRNA - contains a RBS (ribosome binding site ) / also known as a

Shine- Delgaro sequence.

➢ The RBS is characterized by a core sequence 5' AGGAGU 3' locited 7a

2 moleotides from the AUG.
 Translation

Factors required for translation:

A. Prokaryotic
2. tRNA - adapter molecules in the information transfer between mRNA
and protein, which has:

a) anticodon which is a 3 nucleotide sequence that is complementary and

anti-parallel to the mRNA codon

b) amino acid attachment site at the 3' end lox attachment of the amA
Factors required for translation:

c) 3-1) shape that determines which amino acid will be attached to the
amino acid attachment site.

➢ Recent studies indicate that the anticodon loop, the D loop, and the
aminoacyl stem are all important.

➢ The correct attachment of the amino acid to its tRNA is considered

the "Ind genetic code" and is still being cracked.

d) Isoacepters = tRNAs with different anticodons but same amino acid.

e) Aminoacyl tRNA = tRNA with an amino acid attached changed

tRNA,
 Factors required for translation:

3. Aminoacyl tRNA synthetase - transfers the amino acid to its proper tRNA:

➢ There are 20 of these, each recognising 1 amino acid and all the tRNAs that
to which that amino acid is to be attached

4. Ribosomes

a) Large subunit (50s) - consists of 23s and 5s rRNAs and 33 ribosomal

proteins

b) Small Subunit (30s) consists of 16s RNA and 20 ribosomal Proteeins

 Factors required for translation.

Soluble translation factors:

a) Initiation factors

1) IF 1 - promotes dissociation of ribosomal subunits

2) IF2 *(GTP) - required for fMET tRNA met binding

3) IF3 - required for mRNA binding, finding the AUG

Elongation factors

1) EF-Tu *(GTP)-binds amino acid tRNA to the ribosome

2) EF-Ts - regenerates EF-Tu*GTP

3) EF-G*(GTP) - increases translocation rate

5. Soluble translation factors:

c) Termination factors

1.RF1 - recognizes UAA and UAG stop codons

2.RF2 - recognizes UAA and UGA nonsense codons
3.RF3(•GTP) - enhanced RF-1 and -2 binding to ribosome

[Link] acids
[Link]
[Link] (for Charging tRNAs)
Factors required for the translation of Eukaryotic proteins

(similar to prokaryotes except... )

[Link] gene per mRNA (monocistronic)

[Link]

Interestingly, there are only two ribosomal proteins and the rRNA which
are very highly conserved among prokaryotes and eukaryotes.

For eukaryotes,

a) Large subunit (60S)- consists of 28S, 5.8S, and SS rRNAs and S0

ribosomal proteins,

b) Small subunit (40s) consists of 18s rRNA and 33 ribosomal proteins

3. Soluble translation factors:

a) Initiation factors
[Link] 1 - promotes dissociation of ribosomal subunits

[Link] 2"(GTP) - required for fMET tRNAmet binding

3.elF3 -
4.elF4 - important for finding the capped end of the mRNA

b) Elongation factors

[Link] (•GTP) - binds AA-tRNA to the ribosome

[Link] - regenerates EF •GTP

3.EF2(-GTP) - increases translocation rate

c) Termination of several TF (Termination factor)

Important

Translation mechanism in Prokaryotes :

1) Initiation: Prokaryotic Model = the purpose of this step is to set the

reading frame

Formation of the Initiation complex:

➢ Small subunit of ribosome binds with initiation factors (IFs) IF1, IF2,
IF3, and GTP (energy source).

➢ Initiation Complex binds to Shine Delgarno sequence ”5' AGGAGU

3" or "5' AGGAGG 3' "on leader sequence of mRNA (which sets the
reading frame of the initial AUG) and loses IF3
 Translation mechanism in Prokaryotes :

➢ Formation of Initiation complex:

➢ Initiation codon of mRNA binds to fMet IRNA

(formylmethionine tRNA a modified form of fmet tRNA).

➢ The Shine-Delgaro sequence, 5' AGGAGG 3', complements

sequences 3’ NCCUCC 5’ of the 16s rRNA subunit of the
ribosomal small subunit.

➢ Together, these components form the 30s initiation cpmplex

Translation mechanism in Prokaryotes :

➢ Thereafter, attachment of the SOS (large) subunit is facilitated through

dephosphorylation of GTP to GDP and removal of IF I and IF2.

➢ The 30S initiation complex and S0S ribosomal subunit complex together form a
70S initiation complex where fMet IRNA anticodon 3' UAC S' complements

➢ mRNA start codon S' AUG 3' in the P (peptidyl) site of the ribosome.

➢ The ribosome has two sites for binding of aminoacyl-tRNA (or "charged

➢ IRNA") the Peptidyl (or P) site and the Aminoacyl (or A) site
 Translation mechanism in Prokaryotes :

Initiation: Eukaryotic Model

➢ Eukaryotic initiator factor elF-4E, which includes cap binding protein (CBP)

➢ binds to cap of mRNA - recall that prokaryotic mRNA has no cap

➢ 40s ribosomal (small) subunit binds to Met- tRNA (unmodified methionine but a
special initiator form of RNA), additional elFs and GTP, forming a complex that
moves along mRNA until it finds initial start codon, AUG.

➢ Next, this 405 initiation complex binds to the 605 (large) ribosomal subunit with the
help of GTP. and elF's are displaced.

➢ 'This latter complex the 80s initiation complex

Best of Luck!

Fazal Amin