Charging of tRNA, Aminoacyl tRNA Synthetases

Charging of tRNA, Aminoacyl tRNA Synthetases

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  Charging of tRNA,aminoacyl tRNA synthetases Dr.Sujit Ghosh
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  Role of TransferRNA:  Transfer RNA molecules play a key role in the process by delivering amino acids to the ribosome in an order specified by the mRNA sequence; this ensures that the amino acids are joined together in the correct order. Cells usually contain many species of tRNA, each of which binds specifically to one of the species of tRNA, each of which binds specifically to one of the 20 amino acids.  there may be more than one tRNA for each amino acid. Transfer RNAs that bind the same amino acid are called iso-acceptors.
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  What is aminoacylationor charging ?  Before translation begins, amino acids become covalently linked to their tRNAs which then recognize codons in the mRNA specifying that amino acid. The attachment of an amino acid to its tRNA is called amino acylation or charging. The amino acid is covalently attached to the end of the acceptor arm of the tRNA which always ends with the base sequence 5′ CCA 3′.  A bond forms between the carboxyl group of the amino acid and the 3′- hydroxyl of the terminal adenine of the accep­tor arm.  Charging is catalyzed by enzymes called aminoacyi tRNA synthetizes in a  Charging is catalyzed by enzymes called aminoacyi tRNA synthetizes in a reaction requir­ing the hydrolysis of ATP. A separate enzyme exists for each amino acid and each enzyme can charge all the iso-acceptors tRNAs for that amino acid.  The aminoacyi tRNA synthetase recognizes both the appropriate amino acid and the corre­sponding tRNA.  When the correct amino acid has been attached to the tRNA, it recognizes the codon for that amino acid in the mRNA allowing it to place the amino acid in the correct position, as speci­fied by the sequence of the mRNA. This ensures that the amino acid sequence encoded by the mRNA is translated faithfully.
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  Aminoacyl-tRNA synthetases (ARSs) Aminoacyl-tRNA synthetases (ARSs) are highly conserved group of ancient enzymes that are critical components of the cellular protein synthesis machinery . They catalyze the two-step aminoacylation reaction during protein synthesis.  First step involves activation of amino acid by an ATP molecule resulting in the formation of aminoacyl adenylate and release of inorganic pyrophosphate (PPi)  In second step the activated amino acid is transferred to the 2'- or 3'-OH group of the terminal adenosine of its cognate tRNA while releasing AMP  Each ARS catalyzes the covalent attachment of a single amino acid to one or more tRNA isoacceptors to form charged tRNAs. Molecular elements within the tRNAs serve as determinants or anti- determinants that aid in selection by cognate ARSs
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  ARS classification -basedon chemical properties of their primary sequence homologies and architectures of their secondary structures (catalytic domains) Class 1 are mostly monomers Class 2 are typically dimers or are mostly monomers attach the amino acid to the 2’-OH of the tRNA The catalytic site of Class I ARSs comprises of a characteristic Rossmann dinucleotide binding fold marked by HIGH and KMSKS signature peptides dimers or tetramers. attach the amino acid to the 3’-OH of the tRNA possess a more unique catalytic core.
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  tRNA Structure 1. tRNAs areadapters between amino acids and the mRNA molecules. 2. General structure highly conserved in prokaryotes and eukaryotes. 3. Transcribed as larger precursor, processed 1. Bases modified. 2. Modified bases lead to improved tRNA 2. Modified bases lead to improved tRNA function 4. Amino acid is covalently attached to the 3’ end of the tRNA. 5. tRNAs often contain modified nucleosides. 6. tRNAs have complementary sequence to mRNA.The anticodon of the tRNA base pairs with the codon of mRNA.
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  Cloverleaf structure  tRNACloverleaf structure 1.The 5'-terminal phosphate group. 2.The 3' end of the tRNA molecule has CCA tail -important for the recognition of tRNA by enzymes. tRNA by enzymes. - amino acid is attached to A 3.Acceptor stem 4. Has 3 stem loops - D loop (contains dihydrouridine) - Anticodon loop (5’ IGC 3”) -T loop ( contains sequenceTΨC where Ψ is a pseudouridine) 5. Presence of aVariable loop
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  3D structure oftRNA
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  tRNA molecules must havethe correct anticodon sequence. tRNA molecules must be recognized by the correct aminoacyl-tRNA synthetase. tRNA molecules must bind to the appropriate sites on the ribosomes. Specificity of tRNAs sequence. on the ribosomes.
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  Proofereading ARS Kinetic proofereading Chemical proofereading
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  Aminoacylation reaction isan error prone mechanism.There is a possibility of mis-activation and transfer of structurally similar amino acids by a single 1(ii)). If this phenomenon is prevalent in cells, it would lead to mis-incorporation of aminoARS (Figure acids generating defective proteins that might affect cellular viability . In order to ensure fidelity, some of the synthetases have evolved to perform editing functions to reduce the occurrence of errors during protein synthesis.Therefore,ARSs have evolved to perform editing functions to generate higher proportion of accurately synthesized proteins in cells. pre-transfer (correction of mis-activated amino acid) or post-transfer (correction of mis-charged tRNA) of amino acid to the tRNA