Translation In Prokaryotes And Eukaryotes

1. Introduction
2. Messenger RNA (mRNA)
3. Transfer RNA (tRNA)
4. Aminoacyl tRNA synthetase
5. Ribosome
6. Initiation of translation
7. Elongation of translation: Peptidyl transferase reaction
8. Termination of translation
9. Antibiotics: Target and consequences

CHARACTERIZATION OF CELL LINES

1. Introduction

The translation is the process in which a sequence of nucleotide triplets in a messenger RNA gives rise to a specific sequence of amino acids during the synthesis of a polypeptide chain or protein.
The translation is among the most highly conserved across all organisms
Protein synthesis requires the coordinated action of well over 100 proteins and RNAs

Translational machinery

The machinery required for translating the language of messenger RNAs into the language of proteins is composed of four primary components
i. mRNAs
ii. tRNAs
iii. Aminoacyl tRNA synthetase
iv. Ribosome.
Together these components accomplish the extraordinary task of translating the code written in a four base alphabet(A, T, G, C) into a second code written in the language of 20 amino acids

Messenger RNA (mRNA)

Messenger RNA (mRNA) provides an intermediate that carries the copy of a DNA sequence that represents a protein.
Protein coding region of mRNA is composed of a contiguous, non-overlapping string of codons called Open reading frame (ORF).
mRNA containing a single ORF is Monocistronic mRNA.
mRNA containing multiple ORRFs is Polycistronic mRNA.
Codons – ordered series of three nucleotides specific for amino acids.
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Start codon – first codon of an ORF present at the 5’ end

Prokaryotic start codon
5’-AUG-3’
5’-GUG-3’ –
5’-UUG-3’

Eukaryotic start codon

5’-AUG-3’

Stop codon – last codon of an ORF at the 3’ end which defines the signal termination of polypeptide synthesis.

3 stop codons are –

5’-UAG-3’
5’-UGA-3’
5’-UAA-3’

PROKARYOTIC mRNA

Prokaryotic ORFs contain short sequences, (3-9bp) upstream of start codon called Ribosome binding site(RBS) or Shine Dalgarno Sequence.
The core of the sequence is mostly a subset of sequence 5’-AGGAGG-3’ which binds to the 5’-CCU CCU-3’ of 16S rRNA.
Some prokaryotes that lack RBS undergo Translational coupling as they contain overlapping open reading frames.
ANIMAL CELL CULTURE MAINTENANCE

EUKARYOTIC mRNA

3 modifications for the recruitment of ribosome to mRNA
1) Methylated guanine cap at the 5’ end
2) Kozak sequences:- the presence of a purine 3 bp upstream of AUG and presence of guanine immediately downstream (5’-G/ANNAUGG-3’)
3) Presence of a poly A tail at the extreme 3’ end

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TRANSFER RNA (tRNA)

tRNA acts as an adaptor between the codons and the amino acids
they specify.
There are many types of tRNA molecules, but each is attached to a
specific amino acid and each recognizes a particular codon
75-95 ribonucleotides in length
Terminus is 5’-CCA-3’ which is the binding site of amino acid to tRNA
Contains unusual or modified bases- uridine, thymine, pseudouridine, methylguanine, hypoxanthine etc.
They are not essential for tRNA but those lacking these show a reduced rate of growth.

Modified bases in tRNA

Structure of t-RNA

tRNA share a common secondary structure that resembles a
cloverleaf
Acceptor arm – 5’-CCA-3’ which is the binding site of amino acid to tRNA
ΨU loop – contains Pseudouridine (ΨU )
D loop – conations dihydrouridine
Anticodon loop – binds to codons in mRNA
Variable loop – varies from 3 to 21 bases

Biochemistry Satyanarayana Chakrapani

Structure of tRNA

Amino Acyl tRNA Synthetase

Also called charging of tRNA
Catalyzed by Aminoacyl tRNA synthetase
2 classes of aminoacyl tRNA synthetase
Class 1- monomeric which attach amino acid at 2’ OH of tRNA
Class 2- Dimeric or tetrameric which attach aa. at 2’ OH of tRNA

Aminoacyl tRNA synthetase

Attachment of amino acids to tRNA

Amino acids are attached to tRNA in two steps
1) Adenylation – Amino acid with the ATP to become adenylated. the carbonyl group of the amino acid is ionized to the phosphate group of AMP by releasing PPi from ATP.
2) Charging – Carbonyl group of adenylated react with 3’OH of tRNA. A high energy bond and the concomitant release of AMP.

CONTAMINATION IN ANIMAL CELL CULTURE

Attachment of amino acids to tRNA

RIBOSOME

It is the macromolecular complex that directs the synthesis of proteins.
Composed of a large subunit and a small subunit
Large subunit contains the peptidyl transferase center which is responsible for the formation of peptide bonds
Small subunit contains the decoding center in which charged tRNAs read or decode the codons of mRNA
Large and small subunits undergo association and dissociation during each cycle of translation,

ROLE OF rRNA in RIBOSOME

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Ribosomes are made up of 50% RNA and 50% proteins.
RNA in ribosomes are called rRNA which has extensive secondary structures and associates with proteins to form ribosomes.
16 S rRNA in the small subunit of ribosomes is the binding site for the mRNA.
23 S rRNA of the large subunit is the is the ribozyme which catalyzes the peptidyl transferase reaction.
An mRNA bearing multiple ribosomes is known as polyribosome or polysome.

The ribosome has three tRNA binding sites
1) A site – binding site for first aminoacylated tRNA
2) P site – binding site for the peptidyl tRNA
3) E site – binding site for the uncharged tRNA

PRESERVATION OF ANIMAL CELL LINES

These sites are present at the interface between the small and the large subunit of the ribosome

Channels in ribosome

The small subunit of the ribosome has two narrow tunnels
• Entry channel for mRNA
• Exit channel for mRNA
• Large subunit has an exit channel for newly synthesized polypeptide chain,

Initiation Of Translation

It occurs in three steps
1) Ribosome must be recruited to the mRNA
2) Charged tRNA must be placed into the P site of the ribosome
3) Ribosome must be precisely positioned over the start codon
• The initiator tRNA is charged with N-Formyl methionine in prokaryotes and with methionine in eukaryotes.
• Three initiation factors direct the assembly of an initiation complex that contains mRNA and the initiator tRNA called translational initiation factors.

HUMAN HYBRIDOMA

GENERAL OVERVIEW OF INITIATION

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PROCESS OF INITIATION IN PROKARYOTES

IF1 – prevents tRNA from binding to the portion of the small subunit
that will become the part of a site
2. IF2 – a GTPase which interacts with small subunit, IF1, charged initiator tRNA and prevents other charged tRNAs to bind to the small subunit.
3. IF3 – it binds to the small subunit and blocks it from reassociating with a large subunit, or from binding charged tRNA and in the dissociation of 70S ribosome into large subunit and small subunit

Withal three initiation factors bound, the small subunit is prepared to bind to the mRNA and the initiator which can bind in either order.
This results in the formation of the 70S initiation complex

INITIATION IN PROKARYOTES

PROCESS OF INITIATION IN EUKARYOTES

Involves 4 general steps:

Binding of tRNA precedes binding of mRNA
mRNA is recruited separately
Small subunit bound to tRNA scans mRNA for AUG
Large subunit is recruited after RNA base pairs with the start codon,

a. Binding of tRNA to the P site

4 initiation factors are involved – eIF1, eIF1A, eIF2, eIF5.

They bind to the small subunit
eIF1, eIF1A, eIF5 act in an analogous manner to prokaryotic initiation factors of IF3 & IF1 to prevent both large subunit binding and tRNA binding to the A site
RNA is escorted by the GTP binding protein – eIF2
This is Ternary complex
eIF2 positions tRNA at p site

b. Binding of mRNA to the 43S initiation complex

4 initiation Factors involved are eIF4E, eIF4G, eIF4B, eIF4A.
Recognition by 5’ cap by a 3 subunit complex eIF4E
eIF4G binds to eIF4E and mRNA, to which binds eIF4A
It is then joined by eIF4B which activates an RNA helicase activity of eIF4A that unwinds any secondary structure
This eIF4F-eIF4B complex is then recruits the 43S preinitiation complex to the mRNA by interactions between eIF4F and eIF3.
This 43S complex with mRNA is called 48S preinitiation complex.

SYNTHETIC CULTURE MEDIA

PROCESS OF INITIATION IN EUKARYOTES

c. Scanning for AUG
After assembling the 5’end of mRNA, the small subunit scans the mRNA for the start codon in 5’ – 3’ direction in an ATP dependent process
Correct base pairing between the initiator tRNA and start codon releases eIF3 and eIF2 which allows the large subunit to bind to the small subunit.
Binding of large subunit leads to loss of eIF5B by GTP hydrolysis and binding of initiator tRNA to P site and formation of 80S complex

d. Association of the small and large subunit

Correct base pairing changes the conformation of 48 S complex leading to the release of eIF1and a change in conformation of eIF5
Both these events hydrolyze eIF2GTP into eIF2 GDP
Loss of eIF2GDP stimulates the loss of eIF5B which stimulate the correct base pairing of the large and small subunit of the ribosome.

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Peptidyl transferase reaction

Once the correctly charged tRNA has been placed in the A site and has rotated in the peptidyl transferase center, peptide bond formation takes place
This reaction is catalyzed by the 23S r RNA component of the large subunit
So ribosome is called a Ribozyme
Base pairing between the 23S rRNA and the CCA end of tRNA in the A site and the P sites help to position the alpha amino group of the aminoacyl tRNA to attack the carbonyl group of the growing the polypeptide chain attached to the peptidyl tRNA. Role of EF- G
EF-G drives the translocation by displacing the tRNA bound to the A site.
EF-G GTP binds to the factor binding center on the large subunit of the ribosome.
It then dephosphorylates to form EF-G GDP and there is a structural change in it and it binds to mRNA at the A site.
EF-G GDP displaces the A site tRNA to the P site and essentially the mRNA is moved along with this movement.
After translocation, changes in the structure of large subunit allow EF-G GDP to be released.

Role of EF G in Elongation

Termination Of Translation

Stop codons are recognized by proteins called release factors(RF)
These activate the hydrolysis of polypeptides from the peptidyl tRNA.
There are 2 classes of RF:
Class 1 RFs – recognize stop codon and trigger the hydrolysis of peptide chain from the peptidyl tRNA
Class 2 RFs – stimulate the dissociation of class 1 RFs from the ribosome after the release of the polypeptide

RF CLASS

CLASS 1

CLASS 2

PROKARYOTES

RF 1 – UAG

RF 2- UGA

RF `1 & RF 2 – UAA

RF 3

EUKARYOTES

eRF 1- All stop codon

eRF 3

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All class 1 factors share a conserved three amino acid sequence (glycine, glycine, glutamine- GGQ) that is essential for polypeptide release
Studies have led to the hypothesis that class 1 RFs functionally mimic tRNA; having a peptide anticodon that binds to the stop codon and GGQ motif reaches the peptidyl transferase center
Steps involved are;
1) After RFs bind to the A site and recognize stop codon, there is a conformational change in RF which releases the polypeptide
2) RF 3 GDP binds on class 1 RFs after the release of the polypeptide
3) Change in the conformation of ribosome and RFs stimulates RF3 to exchange its bound GDP to GTP
4) This RF3 GTP forms a high-affinity interaction with ribosome that displaces class 1 RFs and concurrent hydrolysis of GTP into GDP
5) Now this RF- GDP has a low affinity for ribosome and is released

TERMINATION OF TRANSLATION IN PROKARYOTES

TERMINATION OF TRANSLATION IN EUKARYOTES

Ribosome Recycling

After the release of the polypeptide and the release factors, the ribosome is still bound to the mRNA and is left with two deacylated tRNA (in the P and E sites).
• To participate in a new round of polypeptide synthesis, these mRNA and the tRNA must be released and the ribosome must dissociate into small subunit and a large subunit.
• Collectively these events are termed as ribosome recycling
• In prokaryotes, a factor called ribosome recycling factor (RRF) cooperates with EF-G and IF3 to recycle ribosome

RRF binds to the empty A site of the ribosome where it mimics tRNA.
• RRF also recruits EF-G to the ribosome which stimulates the release of uncharged tRNA
• After the release of tRNA, IF3 may also participate in the release of the mRNA and is required to separate the two subunits of ribosomes.

RIBOSOME RECYCLING

Antibiotics arrest cell division by blocking the steps of translation

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Antibiotics,mRNA, EUKARYOTES, Scanning, small and large subunit, Elongation in Prokaryotes, Role of EF G in Elongation, TERMINATION OF TRANSLATION IN EUKARYOTES, RIBOSOME RECYCLING,