Protein synthesis

• Types and site of protein synthesis:• 1. body proteins: antibodies, plasma proteins,

contractile proteins in the muscles , all enzymes and some hormones.

• Mammary gland = milk proteins, muscles makes contractile proteins etc.

Requirements for protein synthesis

• Amino acids: • tRNA• mRNA• Aminoacyl –t RNA synthetase enzyme• Ribosome• Protein factors• High energy compounds :ATP and GTP

Steps

• Initiation • 1. it requires mRNA molecule is selected for

translation by ribosomes. Once the mRNA binds to the ribosome ,the later reads the genetic code on the mRNA and translation begins.

Requirements for initiation process

• tRNA • Ribosomes• mRNA• Amino acids• GTP and ATP• At least 10 eukaryotes initiation factors (eIFs)

Initiation can be divided into 4

• Ribosomal dissociation :dissociation of the complete ribosome 80S into its 40S and 60S.

• Step is carried out by two initiation factor (eIF-3 and eIF1A) which binds to 40 S subunit ,dissociating it from 80S and prevent its reassociation.

Formation of 43S preinitiation complex

• Complex is formed by a ternary complex consisting of met-tRNAs,GTP and eIF-2 to the 40S ribosome:

• 1. first: binding of GTP with eIF-2.This binary complex then binds to met –tRNA ;to form a ternary complex.

• 2. ternary complex then binds to the 40S ribosomal subunits to form 43S preinitiation complex.

Formation of 43S initiation complex

• Complex is formed by binding of mRNA to the 43S preinitiation complex.

• 1. initiation factor four subunit (eIF4)interact with mRNA cap.• 2.mRNA cap-together with eIF4-facilitate the binding of

mRNA to the 43S preinitiation complex.• 3.ATP hydrolysis occurs during binding of mRNA.• 4. recognition of initiation codon: AUG at untranslated region

of mRNA.The scanning mechanism starts by binding of 43S preinitiation complex with mRNA neaqr its 5’end.Then it moves 3’end until it meets first AUG codon.ATP hydrolysis is essential for scanning process.

Formation of 80S initiation complex

• Complex is formed by binding of the 48S initiation complex with 60S ribosomal subunit.

• 1. binding involves the hydrolysis of the GTP bound to eIF2 by eIF-5,with release of initiation factors IA and 3.These factors are then recycles.

• 2. complete ribosome 80S contains two sites for tRNA molecules.These are P site (peptide) site which is occupied now by met-tRNA and A (amino) site which is free and ready to receive the subsequent aminoacyl tRNA.

Elongation

• 1. elongation of the polypeptide chain involves the addition of amino acids to the carboxyl end of the growing polypeptide chain.

• 2. during elongation the ribosome moves from the 5’ end to the 3’ end of the mRNA.

Requirements for elongation

• A. 80S initiation complex• B. aminoacyl –tRNA’s• C.GTP• D. Eukaryotic elongation factors,eIF-1 alpha

and eIF-2• Met –tRNA is present in P site(from initiation)

Elongation can be divided into 3 stages.

• Binding of amino acyl tRNA to the A site• A. entry of a new aminoacyl –tRNA to the

empty A site on the ribosome requires proper codon recognition.This depends on the complementary bases of both codon and anticodon.

• At first eEF-a forms a complex with GTP and the entering aminoacyl-tRNA.

Cont.

• This complex allows the aminoacyl –tRNA to enter the A site with the release of eEF-1GTP and GDP and phosphate .These factors are often are then recycled.

• Peptide bond formation:• The carboxyl group of the amnioacyl-tRNA in P site

transferred to and bind with the amino group of the new aminoacyl –t RNA in the A site .This reaction is catalyzed by peptidyl transferase present in 60S subunits.

Translocation

• 1. after the peptide bond has been formed ,the ribosome moves three nucleotides towards the 3’ end mRNA.

• 2. This process is known as translocation and it requires translocation enzyme ,eEF-2 and GTP.

Cont.

• 3. movement of ribosome ,the following events occurs:

• 1. release of uncharged tRNA from P site of ribosome.

• 2. tansfer of the newly formed peptidly tRNA from A site to occupy P site.

• A site becomes free,thus it can be occupied by another new aminoacyl tRNA according to the codon-anticodon recognition.Then process will repeated.

Cont.

• For each new peptide bond formed ,four high energy phosphate bonds are cleaved.

• The charging of the tRNA mol. With aminoacyl group requires the hydrolysis of an ATP to an AMP +ppi .Thus equivalent to the hydrolysis of 2ATP’s to 2 ADP +ppi .

• The entry of aminoacyl tRNA into the A site leads to the hydrolysis of an GTP to GDP + pi

Cont.

• The translocation of the newly formed peptidyl tRNA in the site A site into P site by eEF-2 ,similarly to the hydrolysis of one GTP to GDP +ppi.

termination

• 1.It is the final stop which occurs after multiple cycles of elongation and formation of protein mol.

• 2. it occurs when ribosome moves to bring one of the three termination (nonsense) codons into A site. These are UAA,UAG and UGA.

• Releasing factors (eRF) 1,2 and 3 which are present in A site can recognize all the three termination codons.

Cont.

• Releasing factors together with GTP and peptidyl transferase promote the hydrolysis of the bond between peptide and tRNA occupying the P site.This hydrolysis leads to :

• 1. release of both peptide and tRNA.• 2. dissociation of 80S ribosome into its 40S

and 60S units which are then recycled.

Polyribosomes(polysomes)

• Because the length of the nucleotide sequence of most mRNA s, more than one ribosome can translate the same mRNA at the same time .Such a complex of one mRNA and a number of ribosomes is called a polyribosomes of polysomes.

Post translational modification

• Conversion of inactive protein to active proteins:e.g zymogens,proinsulin.

• Glycosylation: addition of carbohydrates, glycoproteins

• Hydroxylation: addition of OH group, hydroxy proline and hydroxy lysine.

• Phosphorylation: additon of phosphate group,glycogen synthase .

Inhibitors

• Streptomycin. Binds to ribosomes;dissociation of mRNA from the ribosomes.

• Tetracyclins: prevent aminoacyl t RNA asnticodon from recognizing their corresponding codons.

• Chloramphenicol: inhibits peptidyl transferase.

Cont.

• Puromycin: inhibits the elongation• Erythromycin: inhibits the translocation

process.