Translation 2

Translation

Vipin Shankar

mRNA

70s initiation complex

30s initiation complex

Elongation 

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Once the ribosome is assembled with the charged tRNA in the P site, polypeptide synthesis can begin. 3 key events  

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The correct amino acyl tRNA is loaded in the A site. A peptide bond is formed between the amino acyl tRNA in the A site and peptide chain of the peptidyl tRNA in the P site. Translocation of the resulting peptidyl tRNA from A site to P site.

Elongation… 

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As with the original positioning of the mRNA, the shift must occur precisely to maintain the correct reading frame. Two auxiliary proteins, Elongation factors, control these events. Both elongation factors are GTP binding proteins.

Delivery of amino acyl tRNA to A site 

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Amino acyl tRNA is ‘escorted’ to the ribosome by the elongation factor Ef-Tu. Ef-Tu binds to the 3’ end of the tRNA masking the coupled amino acid- this prevents formation of peptide bonds outside the translation assembly. Ef-Tu binds and hydrolyses GTP; the type of Guanine nucleotide bound to it governs its function. Only [Ef-Tu/GTP] can bind to amino acyl tRNA.

Delivery of amino acyl tRNA to A site… 

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Hydrolysis of Ef-Tu bound GTP is triggered by the factor binding center. Ef-Tu comes in contact with the factor binding site only after the correct codon-anti codon match is made. When Ef-Tu hydrolyses its bound GTP, any associated amino acyl tRNA is released.

Selection of correct amino acyl tRNA  

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The error rate of translation is 10-3 to 10-4. The ultimate basis for selection is the base pairing between the charged tRNA and the codon at the A site. But this alone is not responsible for the high rate of accuracy.

Selection of correct amino acyl tRNA.. 

Mechanism 1 

2 adjacent Adenine residues are present in the 16s rRNA of the small subunit.

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These bases form a tight interaction with the minor groove of each correct base pair formed between the anti codon and the first 2 bases of the codon.

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Non-Watson base pairing form a minor groove which is not recognized by the ‘A’ pair, and dissociation of the aa-tRNA takes place.

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Correctly bound aa-tRNA has a decreased dissociation rate.

Selection of correct amino acyl tRNA.. 

Mechanism 2 

The GTPase activity of Ef-Tu depends on codon- anti codon base pairing.

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Even a single mismatch leads to a dramatic reduction in GTPase activity.

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This is an example of kinetic selectivity.

Selection of correct amino acyl tRNA.. 

Mechanism 3 

Occurs after release of Ef-Tu.

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When the charged tRNA is first introduced its 3’ end is distant from the site of peptide bond formation.

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To participate in the peptidyl transferase reaction, the tRNA must rotate into the peptidyl transferase reaction center of the large subunit – Accommodation.

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Incorrectly paired tRNAs cannot bear the strain during accommodation and hence dissociates.

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Referred to as Proof reading.

Formation of the peptide bond 

This reaction is catalyzed by the 23s rRNA of the large subunit.

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The exact mechanism is not yet determined.

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It is proposed that the 23s rRNA base pairs between the CCA ends of the tRNAs at A site and P site.

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This positions the α -amino group of the aatRNA to attack the COO- group of the growing polypeptide on the p-tRNA. These interactions also stabilize the aa-tRNA after accommodation.

Peptide bond formation

Translocation 

Once the peptidyl transferase reaction has occurred, the tRNA in the P site is deacetylated and the growing peptide chain is linked to the tRNA in the A site.

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For a new round of peptide chain elongation, the P site tRNA must move to the E site and the A site tRNA to the P site.

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At the same time the mRNA must move by 3 nucleotides to expose the next codon.

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These movements are coordinated within the ribosome and are collectively referred to as translocation.

Translocation… 

Once the growing peptide chain has been transferred to the A site tRNA, the 3’ end of this tRNA move to the P site in the large subunit, while its anti codon end remains in the A site of the small subunit.

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Similarly, the now deacetylated P site tRNA is located in the E site of the large subunit and the P site of the small subunit.

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Thus translocation in the large subunit precedes that in the small subunit and the tRNAs are said to be in ‘hybrid state’.

Translocation… 

Completion of translocation requires elongation factor Ef-G.

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In the hybrid state the tRNA in the A site uncovers the Ef-G-GTP binding site at the A site of the large subunit.

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When Ef-G-GTP binds, it comes in contact with the factor binding center of the large subunit.

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This triggers the hydrolysis of GTP by Ef-G.

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Ef-G-GDP has a different conformation, which helps it to reach the A site of the small subunit and trigger the translocation of the small subunit.

Translocation… 

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The movement is initiated by the interaction of the Ef-G-GDP with the decoding center of the small subunit. This causes a displacement of the A site tRNA into the P site, with a related movement of the P site tRNA to the E site. During the movement the mRNA is shifted by 3 base pairs. Essentially the mRNA is pulled along with the moving A site tRNA.

GTP exchange 

Both EF-Tu- GDP and EF-G-GDP should be converted to Ef-Tu-GTP and Ef-G-GTP, for continuing elongation.

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EF-G has a lower affinity to GDP, and GDP is released rapidly after hydrolysis.

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Ef-Tu requires a second protein EF-Ts (GTP exchange factor).

Elongation in eukaryotes 

There is no difference between prokaryotic and eukaryotic translation elongation.

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The factors are named differently 

Ef-Tu is called eEF1

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EF-G is called eEF2

Termination 

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Release factors terminate translation in response to stop codons. 2 classes of release factors 

Class 1  

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2 types in prokaryotes, RF1 & RF2. RF1 recognizes UAG & UAA, RF2 recognizes UGA & UAA. Eukaryotes have only eRF1 – recognizes all 3 stop codons

Class 2   

RF3 in pk & eRF3 in ek. Regulated by GTP. Stimulate release of the class 1 factors.

Termination… 

A region of the release factors called – peptide anti codon composed of 3 amino acids recognize the stop codon.

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All factors share a conserved glycine-glycineglutamine (GGQ) motif, essential for the release of the polypeptide chain.

Release of class 1 releasing factors 

Accomplished by RF3.

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RF3 has high affinity to GDP, exist as RF3-GDP in free state.

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RF3-GDP binds to Class 1 RFs.

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Release of the peptide chain initiates conformational change and GDP is exchanged for GTP, forming RF3-GTP.

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This causes a high-affinity interaction within the ribosome causing the release of the class 1 RF.

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Release of RF brings about a conformational change and RF3-GTP comes in contact with the factor binding site.

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GTP is hydrolyzed and RF3-GDP is released.

Ribosome Recycling. 

After the release of the polypeptide chain & the release factor, the ribosome is still bound to the mRNA and is left with 2 deacetylated tRNAs (in the P site and E sites).

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To participate in new round of peptide synthesis, the tRNAs and the mRNA must be removed and the ribosome must dissociate into the large & small subunits – ribosome recycling.

Ribosome recycling… 

Ribosome recycling factor (RRF) cooperates with Ef-G and IF3 to recycle ribosome.

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RRF binds to the empty A site and mimics a tRNA.

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RRF also recruits EF-G to ribosome and, in events that mimic elongation, the tRNAs at P site and E site are released.

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Once the tRNAs are removed, RRF and EF-G are released along with the mRNA.

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IF3 now binds to the small subunit and the ribosome dissociate into the large and small subunits.

Post translational processing 

Final stage of protein synthesis.

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The nascent poly peptide chain is folded and processed into its biologically active form.

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During or after its synthesis, the polypeptide progressively assumes its native conformation by forming hydrogen bonds, van der Waals’ linkages, hydrophobic interactions, hydrophilic interactions etc.

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The linear or one dimensional genetic message on the mRNA is converted to the three dimensional protein.

Post translational processing.. 

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Amino terminal & carboxyl terminal modifications. Loss of signal sequences. Modifications of individual amino acids. Attachment of carbohydrate side chains. Addition of isoprenyl groups. Addition of prosthetic groups. Proteolytic processing. Formation of di-sulfide linkages.

Amino & Carboxyl terminal modifications 

The first aa inserted during translation is fmet (in pk) & met (in ek).

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However, the formyl group, the amino terminal met residue, and often additional amino terminal residues are removed.

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Enzyme deformylase helps in the removal of formyl group.

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Sometimes residues from the carboxyl terminal are also removed.

Loss of signal sequences. 

15 – 20 residues at the amino terminal play a role in directing the protein to the target – these residues are called the signal sequence.

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Such signal sequences are ultimately removed by specific peptidases.

Modifications of individual amino acids 

The OH- groups of certain Ser, Thr and Tyr of some proteins are enzymatically phosphorylated.

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Many proteins contain monomethyl and dimethyl lysine.

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Calmodulin contains a trimethyl lysine at a specific location.

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In some proteins the COO- of Glu undergo methylation.

Glycosylation 

Attachment of carbohydrate side chains.

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Glycosylation plays an indispensable role in the sorting and distribution of proteins.

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Polysaccharides linked to the amide nitrogen of Asn confers stability on some glyco-proteins.

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Glycosylation plays an important role in cell -cell adhesion.

Glycosylation… 

In some glycoproteins, the carbohydrate side chain is attached enzymatically to Asn residues (N-linked oligosaccharides),

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In others to Ser or Thr residues (O-linked oligosaccharides)

Addition of isoprenyl groups 

A thio-ether bond is formed between the isoprenyl group and a Cys residue of the protein.

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The isoprenyl groups are derived from pyrophosphorylated intermediates of the cholesterol biosynthetic pathway such as farnesyl pyrophosphate.

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Proteins modified in this way include the Ras proteins, products of the ras oncogenes and protooncogenes, and G proteins, and lamins, proteins found in the nuclear matrix.

Addition of isoprenyl groups.. 

The isoprenyl group helps to anchor the protein in a membrane.

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The transforming (carcinogenic) activity of the ras oncogene is lost when isoprenylation of the Ras protein is blocked, a finding that has stimulated interest in identifying inhibitors of this posttranslational modification pathway for use in cancer chemotherapy.

Addition of prosthetic groups 

Many prokaryotic and eukaryotic proteins require for their activity covalently bound prosthetic groups.

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Two examples are the biotin molecule of acetylCoA carboxylase and the heme group of hemoglobin or cytochrome c.

Proteolytic processing 

Many proteins are initially synthesized as large, inactive precursor polypeptides that are proteolytically trimmed to form their smaller, active forms.

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Examples include pro-insulin, some viral proteins, and proteases such as chymotrypsinogen and trypsinogen

Formation of disulfide bridges 

After folding into their native conformations, some proteins form intra-chain or inter-chain disulfide bridges between Cys residues.

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In eukaryotes, disulfide bonds are common in proteins to be exported from cells.

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The cross-links formed in this way help to protect the native conformation of the protein molecule from denaturation in the extracellular environment, which can differ greatly from intracellular conditions and is generally oxidizing.

Inhibition of translation 

Protein synthesis is a central function in cellular physiology and is the primary target of many naturally occurring antibiotics and toxins.

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Natural selection has favoured the evolution of compounds that exploit minor differences in order to affect bacterial systems selectively, such that these biochemical weapons are synthesized by some microorganisms and are extremely toxic to others.

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Because nearly every step in protein synthesis can be specifically inhibited by one antibiotic or another, antibiotics have become valuable tools in the study of protein biosynthesis.

Inhibitors of translation     

Puromycin Tetracycline Chloramphenicol Cyclohexamide Streptomycin

Inhibitors of translation… 

Puromycin 

Made by the mold Streptomyces alboniger.

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Its structure is very similar to the 3’ end of an amino acyl-tRNA, enabling it to bind to the ribosomal A site and participate in peptide bond formation, producing peptidyl-puromycin.

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However, because puromycin resembles only the 3 end of the tRNA, it does not engage in translocation and dissociates from the ribosome shortly after it is linked to the carboxyl terminus of the peptide.

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This prematurely terminates polypeptide synthesis.

Peptidyl transferase

Inhibitors of translation… 

Streptomycin  A basic trisaccharide, causes misreading of the genetic code (in bacteria) at relatively low concentrations and inhibits initiation at higher concentrations.

Antibiotic/Tox in

Target cells

Molecular Target

Consequences

Tetracycline

Pk

A site of 30s subunit

Inhibits amino-acyl tRNA binding to the A site

Hygromycin B

Pk & Ek

Near the A site of 30s subunit

Prevents translocation of A site tRNA to P site

Paromycin

Pk

Chloramphenic ol

Pk

Puromycin

Pk & Ek

Erythromycin Fuisidic acid

Pk Pk

Adjacent to the A Increases error rate of translation site codon-anti by decreasing selectivity of codon codon-anti codon pairing. interaction site inPeptidyl the 30s Blocks correct positioning of the A subunit transferase site amino-acyl tRNA for peptidyl center of 50s transfer reaction. subunit Peptidyl transfer Chain terminator- mimics the 3’ center of large ribosomal subunit Peptide exit

end of amino acyl tRNA in A site and acts as acceptor for the nascent polypeptide chain. Blocks exit of the growing

tunnel of 50s

polypeptide chain from the ribosome; arrests translation. Prevents release of Ef-G-GDP from the ribosome.

Ef-G

Antibiotic/Tox in

Target cells

Molecular Target

Consequences

Thiostrepton

Pk

Kirromycin

Pk & Ek Pk & Ek

Factor binding center of 50s subunit Ef-Tu

Interferes with the association of IF2 and EF-G with the factor binding center. Prevents Ef-Tu release.

Chemically modifies the RNA in the factor binding center of theChemically large subunit

Prevents the activation of translation factor GTPase.

Ricin and αSarcin (Protein toxins) Diptheria toxin

Ek

Inhibits EF-Tu function.

modifies Ef-Tu Cyclohexamide

Ek

Peptidyl transferase center of the 60s Subunit

Inhibits peptidyl transferase activity.