TRNA amino acid attachment

A tRNA molecule is specific for a particular amino acid, though there may be several different forms for each amino acid. Although relatively small, the polynucleotide chain may show several loops or arms because of base pairing along the chain. One arm always ends in the sequence cytosine-cytosine-adenosine. The 3 -hydroxyl of this terminal adenosine unit is used to attach the amino acid via an ester linkage. However, it is now a section of the nucleotide sequence that identifies the tRNA-amino acid combination, and not the amino acid itself. A loop in the RNA molecule contains a specific sequence of bases, termed an anticodon, and this sequence allows the tRNA to bind to a complementary sequence of bases, a codon, on mRNA. The synthesis of a protein from the message carried in mRNA is called translation, and a simplified representation of the process as characterized in the bacterium Escherichia coli is shown below. 

Proofreading by Aminoacyl-tRNA Synthetases The amino-acylation of tRNA accomplishes two ends (1) activation of an amino acid for peptide bond formation and (2) attachment of the amino acid to an adaptor tRNA that ensures appropriate placement of the amino acid in a growing polypeptide. The identity of the amino acid attached to a tRNA is not checked on the ribosome, so attachment of the correct amino acid to the tRNA is essential to the fidelity of protein synthesis. 

Amino acid attachment site Each tRNA molecule has an attachment site for a specific amino acid at its 3 -end (Figure 31.6). The carboxyl group of the amino acid is in an ester linkage with the 3-hydroxyl of the ribose moiety of the adenosine nucleotide at the 3 -end of the tRNA. [Note When a tRNA has a covalently attached amino acid, it is said to be charged when tRNA is not bound to an amino acid, it is described as being uncharged.] The amino acid that is attached to the tRNA molecule is said to be activated. 

Translation involves transfer RNA. (a) The structure of a transfer RNA molecule, with an anticodon at one end and an amino acid attachment site at the other end. (b) A highly simplified symbol for tRNA. The anticodon is a series of three nucleotides that complement the mRNA codon and code for a specific amino acid at the amino acid attachment site. 

Two enzymes are known to add ribonucleotides post-transcriptionally to the 3 hydroxyl end of specific RNAs. One adds the CCA sequence found in all tRNAs at their 3 ends. The 3 terminal adenine in this sequence serves as the amino acid attachment site. The 3 -CCA is relatively unstable and is continually being rejuvenated by this enzyme,... 

The replacement of thymine by uracil has no significant effect on the hydrogen bonding, as RNA does not use base pairing to form complementary dimers it is of less importance than it would be for DNA, but the removal of the methyl group may have an influence on the tertiary structures that RNA can adopt. From this it is clear that DNA is a better method of storing information whereas RNA is more suited to turn that information into a protein sequence. This is done by the ribosome, composed of ribosomal RNA (rRNA), which translates the codons of the mRNA sequence into a protein by matching three base sequences to those of tRNA that have the appropriate amino acids attached. 

At the ribosome, which travels along the mRNA, the tRNA molecule is bound such that its anticodon can interact with a nucleotide triplet on mRNA (the codon). If the anticodon is complementary to a codon triplet on the mRNA, the amino acid attached at the 3 -terminus of the tRNA is transferred to the amino terminus of the growing polypeptide chain if it is not complementanty, the tRNA is rejected and another one is checked for complementary. The whole process is repeated until the synthesis of the protein is completed. It is initiated, as well as terminated, by specific codons regulating this translation. 

The codons of messenger RNA recognize the anticodons of trans-fer RNAs rather than the amino acids attached to the tRNAs. A codon on mRNA forms base pairs with the anticodon of the tRNA. Some tRNAs are recognized by more than one codon because pairing of the third base of a codon is less crucial than that of the other two (the wobble mechanism). 

Structure of a tRNA molecule. The tRNA is a roughly cloverleaf-shaped molecule containing an anticodon triplet on one "ieaf" and an amino acid attached covalently at its 3 end. The example shown is a yeast tRNA that codes for phenylalanine. The nucleotides not specifically identified are chemically modified analogs of the four standard nucleotides. 

Robert Holley first determined the base sequence of a tRNA molecule in 1965, as the culmination ul 7 years of effort, Indeed, his study of yeast alanyl-tRNA provided the first complete sequence of any nucleic acid. This adapter molecule is a single chain of 76 ribonucleotides (Figure 30.2). The 5 terminus is phosphorylated (pCi), whereas the 3 terminus has a free hydroxyl group. T he amino acid-attachment site is the 3 -hydroxyl group of the adenosine residue at the 3 terminus of the molecule. The sequence 5 - IGC-3 in the middle of the molecule is the anticodon, where I is the purine base inosine. It is complementary to 5 -GCC-3, one of the codons for alanine. 

On the basis of the mechanism described on page 871, the base-pairing interaction between the anticodon on the incoming tRNA and the codon in the A site on mRNA determines which amino acid is added to the polypeptide chain. Does the amino acid attached to the tRNA play any role in this process This question was answered in the following way. First, cysteine was attached to its cognate tRNA. The attached cysteine unit was then converted into alanine by removing the sulfor atom from the side chain in cysteine in a reaction catalyzed by Raney nickel the reaction removed... 

The answer is e. (Murray, pp 452—467. Sciivei pp 3—45. Sack, pp 1—40. Wilson, pp 101-120.) For transfer RNAs, the 5 end is often guanosine and is always phosphorylated, while the 3 end is CCA. Although transfer (t) RNA molecules have many features in common, the primary feature that sets them apart is their specificity for different amino acids and the corresponding specific differences of their anticodons. Each tRNA is an L-shaped single chain composed of up to 93 ribonucleotides. Each contains up to 15 methylated bases, and about half of the nucleotides are base-paired into double helices. Activated amino acids attach to the terminal 3 -hydroxyl group of the adenosine. 

When codon-anticodon base pairing occurs the amino acid attached to the tRNA is correctly positioned within the ribosome for peptide bond formation. As each peptide bond is formed, the newly incorporated amino acid is released from its tRNA and the mRNA moves relative to the ribosome so that a new codon enters the catalytic site. The latter process is called translocation. Translation continues one codon at a time until a special base sequence, called a termination or stop codon, is reached. The polypeptide is then released from the ribosome, and folds into its biologically active conformation. Depending on the type of polypeptide, it may then bind to other folded polypeptides to form larger complexes. 

A peptide bond is then formed in a reaction catalyzed by peptidyl transferase, which is a part of the SOS subunit (Step 3). The mechanism for this reaction is shown in Figure 12.13. The a-amino group of the amino acid in the A site performs a nucleophilic attack on the carbonyl group of the amino acid linked to the tRNA in the P site. There is now a dipeptidyl-tRNA at the A site and a tRNA with no amino acid attached (an uncharged tRNA ) at the P site. 

Draw the cloverleaj structure of a tRNA and identify the regions containing the anticodon and the amino acid attachment site. 

Transfer RNA (tRNA) carries amino acids in an activated form to the ribosome for peptide bond formation, in a sequence determined by the mRNA template. There is at least one fRNA for each of the 20 amino acids. Transfer RNA molecules are relatively small as nucleic acids go, with about 70 to 90 nucleotide units. Each fRNA has a three-base sequence, C—C—A, at the 3 hydroxyl end, where the amino acid is attached as an ester. Each fRNA also has an anticodon loop quite remote from the amino acid attachment site. This loop contains seven nucleotides, the middle three of which are complementary to the three-base code word on the mRNA for that particular amino acid. 

The larger subunit becomes attached to form the complete ribosome, which is then ready to accept the next tRNA-amino acid complex at codon n +1 (Phase 2). The insertion of each amino acid residue requires the expenditure of one high-energy phosphate bond, as GTP. 

By means of the recognition site for the amino-acyl-tRNA synthetase the tRNA selects from a mixture of different amino acids the one for whose transfer the tRNA is designed. For example, a tRNA for serine interacts at this site with a synthetase which has bound and activated a serine molecule. What this recognition site looks like in detail and how it functions are still matters of conjecture. In any case, once correctly recognized, the amino acid is transferred to the amino acid attachment region. The amino acid attachment site of a tRNA molecule is, in every case, the -CCA end mentioned above. 

Transfer RNAs typically contain approximately 76 nucleotides and have a molecular mass of about 25 kD. The characteristic cloverleaf secondary structure representation of tRNA was predicted based on regions of base complementarity, and the determination of hundreds of tRNA sequences demonstrated that such a folding pattern is conserved. Several sequence and structural features are also consistently present in tRNAs from all organisms. The sequence at the 3 -end of tRNAs is always -CCA, with a free hydroxyl group on the terminal adenosine that is the site of amino acid attachment. Nucleotides near the termini hybridize to make the 7-base-pair (bp) acceptor stem. 

The two portions of the L can be considered distinct domains with separate contributions to protein synthesis. The minihelix containing the acceptor arm includes the site of amino acid attachment (the 3 -OH). It is considered by many investigators to be related to the historical or early form of tRNA. The anticodon trinucleotide is located at the other end of the L, approximately 75 A away. This... 

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