Cognate tRNAs

Figure 5 Kinetic mechanism of aminoacyl-tRNA selection by the ribosome. The aminoacyl-tRNAs are delivered to the ribosome in the form of a ternary complex with EF-Tu-GTP. Incorrect aminoacyl-tRNAs can either dissociate as a ternary complex in the initial selection phase or later as free aminoacyl-tRNA in the proofreading phase. The two selection phases are separated through the irreversible GTP hydrolysis by EF-Tu. Discrimination against incorrect tRNAs is achieved by increased dissociation rate constants (/r 2 and kj) as well as decreased forward rate constants (ks and ks) compared to cognate tRNAs.

For most amino acids, the ester linkage between the ct-COOH group of the amino acid and the 3 -terminal adenosine of a cognate tRNA is formed in a two-step mechanism catalyzed by an aminoacyl-tRNA synthetase (aaRS). ° In this so-called direct pathway, the aaRS first catalyzes the reaction of the amino acid with adenosine triphosphate (ATP), yielding the enzyme-bound high-energy intermediate aa AMP and PPi in the second step, this aaRS-bound intermediate reacts with tRNA to yield aa-tRNA and AMP (Figure 1). 

GluRS has the characteristic, which is also common to Gin, Arg, and an unusual LysRS, of requiring the presence of its cognate tRNA to catalyze the activation of its amino acid substrate (Figure 1, Section 5.14.1). The crystal structures of complexes of T. thermophilus GluRS with ligand 8 or 9 have been solved, providing an... 

The amino add is linked to its cognate tRNA with an energy-rich bond. 

Step One is the specific attachment of an amino acid to its cognate tRNA... 

In order that tRNA molecules can function as adaptors between amino acids and mRNA, two things must be true. First, there must be a mechanism for attaching each amino acid specifically to its cognate tRNA. Doing so will create molecules known as aminoacyl-tRNAs. Secondly, each aminoacyl-tRNA must recognize the correct triplet code for that amino acid on mRNA. 1 am going to worry about the former issue first. 

There is a family of enzymes that catalyze the attachment of amino acids to then-cognate tRNAs, aminoacyl-tRNA synthetases. There is one or more of these enzymes for each of the 20 amino acids that occur commonly in proteins. Each of these enzymes recognizes (a) a specific amino acid and (b) its cognate tRNA. Imagine a soup of 20 amino acids and 20 tRNAs, one for each amino acid. For example, the aminoacyl-tRNA synthetase for, saline would specifically pick valine out of the soup and catalyze its attachment to the tRNA for valine, tRNA . Simply, we can write the product of the reaction as val-tRNA . This is a lovely example of the role of molecular recognition in a critical life process. 

That driving force is hydrolysis of ATP. The attachment of amino acids to their cognate tRNAs is highly specific. 

Now that we have specifically attached each amino acid to its cognate tRNA, we need to make the interface between each aminoacyl-tRNA and the correct triplet codon on mRNA. The aminoacyl moiety has nothing to do with this it is simply a matter of codon-anticodon recognition, which is shown schematically in figure 13.3. 

T. thermophilus 30S ribosome Cognate tRNA-mRNA IIBM 3.31 1... 

T. thermophilus 30S ribosome Near-cognate tRNA-mRNA Paromomycin IFJG 3.00 13... 

That aminoglycoside binding interferes with translation has been known for at least four decades. The mode of action of these antibiotics decreases the fidelity of translation, such that the organism biosynthesizes proteins that are defective. The presence of defective or nonfunctional proteins leads to the demise of bacteria. Consequently, paromomycin s bactericidal mode of action may come from its ability to lock the A-site into a conformation in which discrimination of cognate and near cognate tRNA is significantly diminished. 

While readthrough is usually a detrimental process, in some cases it can help to suppress problems, e.g. arising from premature stop codons present on the DNA level. This type of readthrough, also termed nonsense suppression, leads to the generation of a fraction of the full length protein in addition to the shortened version. Omnipotent suppressors cause nonsense suppression of all three termination codons. In this process, a near cognate tRNA successfully competes with the termination factors such that amino acid incorporation rather than premature termination of translation occurs. Omnipotent suppression can be caused by mutations in various factors involved in the process of translation termination. Nonsense suppression can also result from an aa-tRNA that decodes a termination codon (suppressor tRNA) in this case only one of the termination codons is efficiently suppressed (Hawthorne and Leupold 1974 Stansfield and Tuite 1994). 

Fig. 4 Schematic section through the small ribosomal subunit of yeast (gray) exposing the decoding region. The model is based on structural work on the prokaryotic small ribosomal subunit. The homologous proteins of the E. coli decoding region are S4, S5, and S12 (Ogle et al. 2003). Movements within the small ribosomal subunit upon cognate tRNA binding (domain closure) are denoted by red arrows. Mutations in...

FIGURE 27-17 Aminoacyl-tRNA synthetases. Both synthetases are complexed with their cognate tRNAs (green stick structures). Bound ATP (red) pinpoints the active site near the end of the aminoacyl arm. 

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