Translation - I

Requirements
 Ribosomal RNA and Ribosomes
What are Ribosomes
- sites/catalyst for protein synthesis.

- facilitating binding of tRNAs carrying aminoacids to the mRNA

so that peptide bonds can form.
 Ribosome Structure

• A cell contains thousands of ribosomes.

• Made of rRNAs and ribosomal proteins.

• Two subunits, large and small.

• Eukaryotes and prokaryotes generally similar but different

enough…
 Prokaryotic Ribosomes
- composed of 65% ribosomal RNA
and 35% ribosomal proteins.

- The unit of measurement is the

Svedberg unit,

- a measure of the rate of

sedimentation in centrifugation
rather than size

- accounts for why fragment names

do not add up (70S is made of 50S
and 30S).
Eukaryotic Ribosomes

60S - 28S rRNA

- 5S rRNA
- 5.8 S rRNA

40S – 18S rRNA

The Nobel Prize in Chemistry 2009

awarded to

1. Drs Venkatraman Ramakrishnan ,

2. Thomas A. Steitz and
3. Ada E. Yonath

"for studies of the structure and function of the

ribosome”
 Ribosome Cycle

In cells, the small and large ribosome subunits associate with

each other and the mRNA, translate it, and then dissociate after
each round of translation.

This sequence of association and dissociation is called the

ribosome cycle
 tRNA Structure

• Adaptor molecule composed of RNA ( 75-90 nucl).

• General structure highly conserved in prokaryotes and

eukaryotes.

• Transcribed as larger precursor, processed

– Bases modified.
– Modified bases lead to improved tRNA function
 tRNA Cloverleaf

1. The 5'-terminal phosphate group.

2. The 3' end of the tRNA molecule has

CCA tail
-important for the recognition
of tRNA by enzymes.
- aminoacid is attached to A

3. - Acceptor stem

4 3 stem loops
a. D loop (contains dihydrouridine)
b. Anticodon loop (5’ IGC 3”)
c. T loop ( contains sequence TΨC where

Ψ is a pseudouridine)

5 Variable loop
3D structure of tRNA
Anticodon

unit made up of three nucleotides that correspond to the three bases of the
codon on the mRNA.

Each tRNA contains a specific anticodon triplet sequence that can base-pair to
one or more codons for an amino acid.

Codon for lysine is AAA.

Anticodon: UUU
Modified Bases in tRNAs
 Attachment of aminoacids to tRNA
2 steps:

1. Adenylation :
- The amino acid react with ATP and becomes adenylated.
- Release of pyrophosphate.

amino acid + ATP → aminoacyl-AMP + PPi

2. tRNA charging :
- adenylated aminoacid transferred to tRNA by
aminoacyltRNA synthetase.

aminoacyl-AMP + tRNA → aminoacyl-tRNA + AMP

Adenylylation

tRNA charging
 tRNA Charging
• Aminoacyl tRNA synthetases
– At least 20 different ones
– One recognizes each amino acid

– Use ATP for energy (AMP + PPi released)

• Adenylated amino acid intermediate
– Carboxyl end of amino acid connected to 3’ end of tRNA
 Isoaccepting tRNAs

• Different tRNA species that bind to alternate

codons for the same amino acid residue.
Two challenges of aminoacyl tRNA synthetases

• Recognize the correct set of tRNAs for a

particular amino acid.
• Charge all of these isoaccepting tRNAs with
the correct amino acid.
Both the processes must be carried out with high
fidelity
• The ribosome has three tRNA-binding sites.

• An aminoacyl-tRNA enters the A site.

• Peptidyl-tRNA is bound in the P site.
• Deacylated tRNA exits via the E site.
 Translation Occurs by Initiation, Elongation,
and Termination

1. initiation – synthesis of the first peptide bond of the

polypeptide, slow step, rate limiting

2. elongation –polypeptide chain is extended by the addition of

individual aminoacids, rapid step

3. termination – release of completed polypeptide chain,

causing disassembly of the synthetic apparatus.

Different accessory factors assist ribosomes at different stage.

Energy is provided by hydrolysis of GTP

 Translation Occurs by Initiation, Elongation,
and Termination
• An amino acid is added to the polypeptide chain by
transferring the polypeptide from peptidyl-tRNA in the P site
to aminoacyl-tRNA in the A site (translocation).
For successful initiation of Translation

3 events must occur:

1. Ribosomes must be recruited to mRNA

2. Charged tRNA must be placed in the P site of ribosomes.

3. The ribosomes must be precisely positioned over the start codon.

 Prokaryotic Translation Initiation

Starts with the formation of the initiation complex involving

1. mRNA strand
2. the small ribosomal subunit (30S)
3. an initiator tRNA
Accessory Factors

3 initiation factors direct the assembly of initiation complex.

1. IF-1
2. IF-2
3. IF-3

- These factors bind only 30S subunit.

- Released when 30S subunit associates with 50S to form 70S.

IF-3 must be released to allow 50S subunits to
join the 30S-mRNA complex.
 Initiation Involves Base Pairing between
mRNA and rRNA

Ribosome-binding site

Signal for initiation – special initiation codon that marks start of

reading frame.
- Usually an AUG
- Can use GUG or UUG (< efficient)
 The AUG is preceded by a Shine-Dalgarno
sequence
Shine–Dalgarno sequence
1. The polypurine sequence AGGAGG
2. centered about 10 bp before the AUG initiation codon on
bacterial mRNA.
3. It is complementary to the sequence at the 3′ end of 16S
rRNA.
 A Special Initiator tRNA Starts the
Polypeptide Chain
• Translation starts with a methionine amino acid usually coded
by AUG.

• Two different methionine tRNAs are involved in initiation and

elongation.
 A Special Initiator tRNA Starts the
Polypeptide Chain

• N-formyl-methionyl-tRNA (tRNAfMet ) – The aminoacyl-tRNA

that initiates bacterial polypeptide translation.
– The amino group of the methionine is formylated.
Initiator tRNA has distinct features

1.Bases at 3’end of acceptor stem are

unpaired in tRNA-fmet

- Needed for formylation.

- Mutations which create base
pairing allow it function in
elongation.

2. 3 GC pairs in anticodon loop unique

to tRNA-fmet.

- required to allow tRNA-fmet to

directly enter the P site
Use of fMet-tRNAf Is Controlled by IF-2 and
the Ribosome

Only fmet-tRNA can enter the partial

P site formed by 30S at start codon –AUG.

Accessory factors control usage of amino-

Acyl tRNAs.
Use of fMet-tRNAf Is Controlled by IF-2
and the Ribosome

IF-2 brings in fmet –tRNA.

IF-3 stabilizes binding of initiator

tRNA by correct base pairing.
 Elongation

involves addition of amino acids to the carboxyl end of the

growing chain.

Elongation starts when the fMet-tRNA enters the P site, causing a

conformational change which opens the A site for the new
aminoacyl-tRNA to bind.

This binding is facilitated by elongation factor-Tu (EF-Tu),

 Three Codons Terminate Translation

• The stop codons UAA (ochre), UAG (amber), and UGA (opal)
terminate translation.

• In bacteria, they are used most often with relative frequencies

UAA>UGA>UAG.