Dr.S.Sethupathy, M.D.,Ph.D.,Professor of Biochemistry,Rajah Muthiah Medical College,Annamalai University

Translation

The process by which the nucleotide sequence of mRNA is converted into the sequence of amino acids of the corresponding protein.

Translation

Synthesis of proteins by ribosomes using mRNA as template is translation

mRNA is the templatetRNA transfers aminoacidsRibosomal RNA worksATP and GTP are required

Translation - animation

Twenty amino acids -proteins At least twenty codes for amino acids. In the protein coding genes, the

nucleotides are organized into a three letter code called codons

The collection of the codons – the genetic code.

Genetic code

The codons of these nucleotides 64(43) specific codons - triplet code.

The nucleotide sequence of the translatable region of an mRNA

Has codons that specify the amino acid sequence of a protein.

Triplet codons - mRNA

There are multiple codons .. Eg; GGA,GGU,GGG,GGC - Glycine methionine and tryptophan have a

single codon One codon represents only one amino

acid One amino acid can have many

codons

Degeneracy

For a given codon in the mRNA, only a single species of tRNA molecule possesses the proper anticodon.

Each tRNA molecule can be charged with only one specific amino acid.

So each codon specifies only one amino acid.

Unambiguous

The genetic code is continuous Non- overlapping There is no punctuation between codons.

The message is read up to a stop codon .

Non-overlapping

Genetic code is universal. Exceptions are: The stop codons are UAA, UGA and UAG.

But in mitochondria AGA, AGG are stop codons.

AUA (Ile) is read as methionine and UGA codes for Tryptophan in mitochondria.

So mitochondria require 22 tRNA species to read their genetic code whereas cytoplasm contains 31 tRNA species.

Universal

The reduced stringency between the third base of the codon and the complementary nucleotide in the anticodon in the tRNA is called wobbling.

e.g: GGU GGC, GGA ,GGG are the codes for glycine. All can pair with CCI (I=inosinic acid) anticodon of tRNA.

Mutations reduced

Wobbling

AUG is the initiator codon. AUG also codes for methionine.

In a few cases, CUG acts as initiator codon.

Initiator codon

Protein synthesis occurs in Ribosomes. tRNA is read from 3’ to 5’ direction Peptide synthesis is from 5’ to 3’

direction. First is N-terminal amino acid Elongation towards carboxy terminal AA.

Reading tRNA & mRNA

The ribothymidine pseudouridine cytidine (TψC) arm is involved in binding of the amino acyl tRNA to the ribosomal surface

At the site of protein synthesis.

TψC arm

The D arm - for recognition of a given tRNA by the specific amino acyl tRNA synthetase enzyme.

D arm

The anticodon region seven nucleotides -3’- variable base- modified purine-

XYZ- pyrimidine- pyrimidine - 5’.. The sequence in tRNA is read from 3’-

5’ direction. tRNA reads the codon in mRNA from 5’

to 3’ direction. - antiparallel.

Anticodon arm

There is no affinity of nucleic acids for specific amino acids,

The recognition is by a protein molecule

It can recognize a specific tRNA and a specific amino acid.

Charging tRNA with AA

Amino acid + Enz +ATP Enz-AM P - Amino acid +ppi

Enz-AMP-Amino acid + tRNA tRNA-AA + Enz +AMP

Enz- Amino acyl tRNA synthetase

Amino acyl tRNA synthetase

Amino acid is attached to 3’-OH –A of CCA sequence of tRNA

Carboxyl group of amino acid – in ester linkage

Incorporated in polypeptide

Amino acid - CCA

1. Initiation2. Elongation3. Termination

Protein Synthesis

1. Dissociation of the ribosome into its 40S and 60S subunits.

2. Binding of a ternary complex consisting of the initiator methionyl tRNA( met-tRNAi), GTP and eIF2 to 40S ribosome to form 43S pre-initiation complex (PIC).

PIC formation

3. Binding of mRNA to the 43S pre-initiation complex to form 48S initation complex.

4. Combination of 48S initation complex with the 60S ribosomal subunit to form 80S initiation complex.

Initiation steps

eIF -3 and eIF-1A delays its re-association of 40S with 60S subunit.

It permits other initiation factors to associate with 40S subunit.

Ribosomal dissociation

eIF is phosphorylated and controls translation.

eIF-2 controls translation

The 5’- cap helps in binding of mRNA to the 43S pre-initiation complex.

A cap binding protein complex eIF-4F which consists of eIF-4E, eIF4G and eIF-4A, binds to the cap through 4E protein.

eIF-4B reduces the complex mRNA structure at 5’-end by its ATP-ase dependent helicase activity.

Formation of the 48S initiation complex

The association of mRNA in the 43S pre-initiation complex requires ATP.

It scans mRNA to find initiation codon.

By KOZAK consensus sequences that surround AUG.( GCC -3A/G, C -1C, AUG, +4G/A).

The presence of purine at positions -3 and +4 relative to AUG is preferred.

Poly(a) tail and its binding protein PAB1 along with eIF 4A, 4E, 4G as a complex binds to the cap.

The binding of 60S and 48S pre-initiation complex to form 80S initiation complex

It involves hydrolysis of GTP bound to eIF-2 by eIF-5.

This releases the initiation factors Association of 40S and 60S subunits to

form 80S ribosome. Now the met-tRNA is on the p site of

ribosome and ready for elongation.

Formation of the 80S initiation complex

The eIF-4F complex controls rate of protein translation.

eIF-4E is required for recognition of mRNA cap, a rate limiting step in translation.

This is regulated by phosphorylation.

Regulation of initiation

Insulin and mitogenic factors phosphorylates 4E on ser 209 or thr 210 and enhance the rate of initiation.

Components of MAP kinase, PI3k, mTOR, RAS and S6 kinases pathways also involved in this phosphorylation.

Proteins such as 4E-BP1, 4E-BP2, 4E-BP3 bind to 4E and inactivates it.

Insulin and growth factors phosphorylate BP1 proteins and prevent its binding to 4E which enhances translation.

Clinical applications

The steps are:1. Binding of amino acyl tRNA to A site 2. Peptide bond formation3. Translocation of ribosome on the mRNA4. Expulsion of deacylated tRNA from P and E sites.

Elongation

Elongation factor 1A (EF1A) forms a ternary complex with GTP and amino acyl tRNA.

EF-1A-GTP-Amino acyl tRNA complex allows the proper amino acyl tRNA to enter A site (Amino acyl or acceptor site) .

EF1A-GDP and phosphate released Elongation factor, EF1B acts (also known

as EF-Ts or EF-1beta/gamma/delta) is involved.

Elongation

The alpha amino group of the amino acyl tRNA in the A site carries out a nucleophilic attack on the esterified carboxyl group of the peptidyl tRNA( P site or peptidyl site).

By a peptidyl transferase , a component of 60S ribosomal subunit. Peptide bond formation

The deacylated tRNA is attached to its codon at p site at one end and open CCA tail to an exit (E) site on the large ribosomal subunit.

Elongation factor 2 (EF2) displaces the peptidyl tRNA from A site to P site and the tRNA on E site leaves the ribosome.

Translocation

EF2-GTP complex is hydroylzed to EF2-GDP and moves mRNA forward by one codon.

Now the A site open for aminoacyl tRNA-EF1A- GTP complex for elongation.

Move to next codon

The stop codon of mRNA appears in the A site.

Releasing factor sits on stop codon in the A site.

RF1 is bound by complex of RF3 and GTP.

This complex with the peptidyl transferase hydrolyze the bond between tRNA in the P site and peptide.

The protein , the tRNA released from the P site.

The 80S ribosome dissociates into its 40S and 60S subunits.

Termination

The charging of tRNAs requires 2ATPs

The entry of amino acyl tRNA into the A site requires 1GTP.

Translocation also requires 1 GTP. So one peptide bond formation requires four ATPs.

ATP for a peptide bond

Many ribosomes can translate the same mRNA molecule simultaneously.

This is called polyribosome or polysome.

Polyribosomes may be free in the cytoplasm or may be attached to endoplasmic reticulum.

Polyribosome

Non-translating mRNAs as ribonucleoprotein particles (RNPs) accumulate in P bodies.

P bodies- decapping enzyme, exonucleases (5’ to 3’ and 3’ to 5’) involved in translation repression or mRNA decay.

certain mRNAs are temporarily stored in P bodies and utilized for translation.

P bodies

When Fe++ is excess, it stimulates the synthesis of ferritin,

by causing a release of cytoplasmic protein that binds to 5’-UTR of ferritin mRNA

It enhances translation.

Clinical applications

poliovirus mRNAs do not have cap structure to direct the binding of the 40S ribosomal unit.

Instead, the 40S ribosomal subunit contacts an internal ribosomal entry site (IRES) which requires 4G and not 4E.

The viral protease removes the amino terminal 4E binding site.

4E-4G complex cannot form and so 40S cannot be directed to the cap of mRNA resulting in the abolition of host cell translation.Clinical applications

The secreted proteins, plasma membrane proteins, lysosomal enzymes and membrane proteins are synthesized on rough endoplasmic reticulum bound polysomes.

These proteins have a signal peptide of about 12-35 amino acids in the amino terminal region.

This signal peptide anchors ribosomes on RER. Signal recognition particle (SRP) is attached

to the signal peptide (SP) region which blocks further protein synthesis.

Protein targeting

SRP-SP ribosome complex binds to a docking protein or receptor (SRPR) on ER.

Now the nascent peptide is passed through membrane into the channels of ER.

The signal peptide is cleaved of by signal peptidase.

Now the protein synthesis is completed and it is within the ER.

Carbohydrate moieties are added to the protein while it transverses the ER and it is called co-translational glycosylation by specific enzymes.

Protein targeting

e.g. KDEL(Lys-Asp-Glu-Leu) amino acid sequence near the C- terminal end is destined to luminal surface of ER.

Proteins destined to peroxisomes contain peroxisome target sequence with 26-36 amino acids.

Nuclear proteins contain nuclear import signal sequences ie: α- importin and β- importin.

Protein address

Zellwager syndromePrimary hyperoxaluria type 1

Cystic fibrosisInclusion cell disease

Clinical applications

cerebrohepatorenal syndrome, is autosomal recessive disorder

characterized by the reduction or absence of functional peroxisomes in the cells

Defective oxidation of very long chain fatty acids .

the proteins are not delivered to peroxisomes. Accumulation of VLCFA causes neurological

impairment, hypomyelination, hepatomegaly, renal cysts, seizures

Patient does not survive beyond one year. Blood VLCFA level is increased.

Zellweger syndrome

Zellweger syndrome- craniofacial abnormalities (such as a high forehead, hypoplastic supraorbital ridges, epicanthal folds, midface hypoplasia, and a large fontanel),

hepatomegaly (enlarged liver), chondrodysplasia punctata (punctate calcification of the cartilage in specific regions of the body),

eye abnormalities, and renal cysts hypotonia (low muscle tone), seizures,

apnea, and an inability to eat

an autosomal recessive disorder due to defective alanine-glyoxylate aminotransferase

Increased excretion of oxalate, with renal and bladder oxalate stones.

The enzyme is found in mitochondria instead of its normal peroxisomal location.

Renal transplant for renal failure and liver transplant for correction of metabolic defect Primary hyperoxaluria Type 1

CF Or Mucoviscidosis is an autosomal recessive genetic disorder that affects the lungs, the pancreas, liver, and intestine.

Abnormal transport of chloride and sodium across epithelium, leading to thick, viscous secretions.

Scarring (Fibrosis) and cyst formation in the pancreas, frequent lung infections, sinus infections, poor growth, salty

tasting skin and infertility are the features. Due to mutation, the protein does not fold correctly and

leads to degradation in the endoplasmic reticulum. The protein never reaches the cell surface.

Lung transplant and gene therapy can be done.

Cystic fibrosis (CF)

Or mucolipidosis II (ML II) is a lysosomal storage disease defective phosphotransferase (an enzyme of the Golgi apparatus). This enzyme transfers phosphate to mannose residues on specific

proteins, and serves as a marker for them to be targeted to lysosomes

they are instead excreted outside the cell. Lysosomes cannot degrade substances (e.g. oligosaccharides,

lipids, and glycosaminoglycans) buildup of these substances- "I cells," or "inclusion cells." lysosomal enzymes instead found in high in the blood. Failure to thrive and developmental delays . Abnormal skeletal

development , coarse facial features, hepatomegaly, splenomegaly, clouding on the cornea of their eyes, trunk dwarfism, recurrent respiratory tract infections .

die before their seventh year of life.

Inclusion cell disease

Reversible Modifications: Disulfide bridge , glycosylation ,

phosphorylation, acylation, N-acylation and ADP-ribosylation .

Irreversible modifications Proteolysis, ubiquitination, lysine,

proline hydroxylation, carboxylation, methylation .

Post translational processing

Gamma carboxylation of glutamic residue of prothrombin

Proteolytic cleavage- pro-insulin to insuln Lysine hydroxylation of collagen -Vitamin

C Phosphorylation of glycogen synthase Glycosylation of proteins- co-translational

or post .T.M.

PTM of proteins

Protein folding A newly synthesized protein may

assume different three dimensional structures.

Chaperones produce the proper and functional spatial arrangements.

Abnormal folding of proteins may lead to prion diseases.

e.g. Heat shock proteins or stress proteins are examples for chaperones.

Chaperones

Defective chaperones - Chaperonopathies. Prion-related illnesses are Creutzfeldt-Jakob disease,

bovine spongiform encephalopathy (mad cow disease), amyloid-related illnesses such as Alzheimer's disease and familial amyloid cardiomyopathy, polyneuropathy, Huntington's and Parkinson's disease.

Degenerative diseases - aggregation of misfolded proteins into insoluble, extracellular aggregates and/or intracellular inclusions.

Misfolding and excessive degradation with loss of function leads to antitrypsin-associated emphysema, cystic fibrosis and the lysosomal storage diseases.

Pharmaceutical chaperones to fold mutated proteins to render them functional.

Chaperonopathies.

Tetracycline binds to 30S subunit of the ribosome and prevents the binding of amino acyl tRNA to bacterial ribosome A site.

Chloramphenicol binds to 50S subunit of the ribosome and inhibits the peptidyl transferase activity of 23S RNA. bacteriostatic and reversible.

Streptomycin is bactericidal and irreversible inhibitor. It binds to 30S subunit of bacterial ribosome, causes misreading of mRNA and inhibits protein synthesis.

Inhibitors of protein synthesis

Puromycin, a structural analog of tyrosinyl- tRNA, binds to A site and causes the premature release of peptide in eukaryotic cells.

Cycloheximide inhibits peptidyl transferase in the 60S ribosomal subunit in eukaryotes and these twoare not clinically useful.

Diphtheria toxin, an exotoxin of Corynebacterium diphtheriae catalyzes the ADP- ribosylation of EF2 and inactivates it in mammalian cells.

Inhibitors

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