Translation

Translation

Objectives

At the end of this lecture, students will be able to –

• Describe the steps involved in the process of translation

• Explain post translational modifications

Content

• Translation process

• Post translational modifications

Translation

• The processed RNA molecule take part in protein synthesis with the help of ribosomes.

• All the three types of RNAs are involved protein synthesis

• Three steps in protein synthesis

1. Initiation of the protein synthesis

2. Elongation of the protein synthesis

3. Chain termination

Initiation in prokaryotes

• The essential factors required for initiation are initiation factors, ribosome, mRNA, GTP, aminoacyl tRNA

• For initiation requires initiation factors like ІF 1, IF2, IF3

• IF1 facilitates the attachment of the 30s subunit to the mRNA and prevent the aa-tRNA from entering the wrong site on the ribosome

• IF2 is a GTP binding protein required for attachment of first aminoacyl- tRNA

• IF3 may prevent the large subunit 50s from joining prematurely to 30s subunit

Binding the first aa-tRNA into ribosomes:

N- formyl methionine is the starting material . Aminoacylated initiator t RNA forms the preinitiation complex by binding to the AUG codon of the mRNA and IF2 initiation factor is released

Assembling the complete initiation complex:

Once the initiator tRNA is bound to the AUG codon, the large subunit joins the complex and GTP bound to the IF2 is hydrolyzed and released

Elongation

• At the end of initiation sequence the 70s ribosome possesses the fmet-tRNA in the p site, where as the A site is free to receive the next aminoacyl- tRNA according to the codon on mRNA

• The elongation factor (EF) is a soluble protein which is required for elongation of peptides

• two types of EF-T and EF-G

• EF-T is associated with tranferase activity( EF-Tu and EF-Ts)

• EF-G is involved in traslocation of mRNA

There are 3 steps in elongation

1. Binding of aminoacyl tRNA to A site

2. Peptide bond formation

3. Translocation.

Binding of aminoacyl tRNA to A site:

• 70s initiation complex contains fmet tRNA in the P site and A site is free

• Before the second aminoacyl tRNA is placed in the A site it must combine with the protein EF linked to the GTP this EF is called as EF-Tu Once the proper t-RNA Tu GTP is bound to the mRNA codon , GTP is hydrolysed and Tu- GDP complex released , leaving the aa- tRNA in the A site

Peptide bond formation

• Enzyme peptidyl transferase catalyzes the formation of peptide bond

• The net result of peptide bond formation is attachment of growing peptide chain to tRNA in A site

Traslocation

• As peptide bond occurs, the ribosomes moves to the next codon of mRNA this is called translocation

• Involves the movement of growing peptide chain from A site to P site

• Requires EF-G and GTP. GTP gets hydrolyzed and supplies energy to move mRNA

Termination

• After several cycles of elongation, incorporating the amino acid and the formation of specific protein / polypeptide molecule, one of the stop codon (UAA, UGA, and UAG) terminates the growing peptide

• Termination codon occupies ribosomal A site, the release factor (RF1, RF2, RF3) recognizes the stop signal

• RF1 is active with UAA and UAG codons RF2 is active with UAA and UGA. RF3 activates the RF1 and RF2 i.e it is called stimulatory factor

• Ribosome binds either with RF1 or RF2 activates the peptidyl transferase which hydrolyze the bond joining the peptide to the tRNA at the P site this result in release of the peptide chain

Initiation in eukaryotes

• It requires 10 initiation factors out of which eIF1, eIF2, eIF3 binds to the 40s subunit, which prepares the subunit for binding to mRNA

• The initiator tRNA (methionine) also binds to 40s subunit prior to its interaction with mRNA

• tRNA enters in association with eIF2 GTP

• This complex is called 43s preinitiation complex

• eIF4E binds to the 5ꞌcap of eukaryotic mRNA

• eIF4A moves along the 5ꞌend of the message removing any double stranded region that would interfere with the movement of 43s complex

• Once the 43s complex binds to the 5ꞌ end of the mRNA, the complex scans along the message until it reaches a recognizable sequence of nucleotide (typically 5ꞌ CCACCAUGC- 3ꞌ) that contains AUG initiator codon

• Once it reaches the AUG eIF2 – GTP is hydrolyzed and released the large subunit 60s joins the complex to complete intiation

Post – translational modification

The protein synthesized in translation are as such not functional

Many changes takes place in polypeptides after the protein synthesis

1. Protein folding

2. Proteolytic degradation

3. Intein splicing

Protein folding:the three dimensional conformation of proteins is important for their biological functions

Chaperons are heat shock proteins facilitate and favour the interaction on polypeptides surface to give the specific conformation of protein

• Chaperons reversibly bind to the hydrophobic region of unfolded proteins and Folding intermediates which helps the protein to attain compact and biologically active conformation

Proteolytic degradation:

• Many proteins are synthesized as precursors which are bigger than the functional protein

• Some portion of precursor molecules are Removed by proteolysis to liberate active proteins

• This process is called as Trimming

Intein splicing:

• Inteins are intervening sequence in certain proteins

• These are comparable to introns in mRNA

• Inteins have to be removed and exteins ligated in appropriate order for protein to become active

Summary

• Translation involves the conversion of mRNA into proteins

• Steps involved are initiation, elongation and translation

• The essential factors required for initiation are initiation factors, ribosome, mRNA, GTP, aminoacyl tRNA