How Synthetases Identify the Correct tRNA Molecule
A second problem in specificity of protein synthesis is the selection of the tRNA molecule by the synthetase. In principle this selection could be done by reading the anticodon of the tRNA. A wide variety of experiments have revealed, however, that only for tRNAMet is the anti-codon the sole determinant of the charging specificity. For about half of the tRNAs, the anticodon is involved in the recognition process, but it is not the sole determinant. For the remaining half of the tRNAs, the anticodon is not involved at all.
Two extreme possibilities exist for the other charging specificity determinants. They could be the identity of one or more nucleotides somewhere in the tRNA. On the other hand, the charging specificity could be determined by part or all of the overall structure of the tRNA molecule. Of course, this structure is determined by the nucleotide sequence, but the structure as dictated by the overall sequence may be more important than the chemical identity of just a couple of amino or carboxyl groups. In view of the diversity of nature, it is reasonable to expect different aminoacyl-tRNA synthetases will utilize different struc-tural details in their identification of their cognate tRNA molecules.
Just as in the study of RNA splicing, the development of genetic engineering has greatly accelerated the rate of progress in under-standing the specificity determinants on tRNA molecules. This resulted from facilitating the synthesis in vitro of tRNA molecules of any desired sequence. Such a synthesis utilizes the phage T7 RNA polymerase to initiate transcription from a T7 promoter that can be placed near the end of a DNA molecule (Fig. 7.5). Essentially any DNA sequence down-stream from the promoter can be used so that any tRNA sequence can be synthesized. The RNA molecules resulting from such reactions can
Figure 7.5 In vitrosynthesis ofan artificial tRNA molecule using T7 RNA polymerase.
be aminoacylated and utilized in translation despite the fact that they lack the specialized chemical modifications that are found on tRNA molecules synthesized in vivo. Apparently these modifications are not essential to the process of protein synthesis and they exist more for fine-tuning.
Figure 7.6 The positions ofnucleotides that determine the charging identity of sev-eral tRNAs.
Genetic engineering also enables us to alter the gene encoding a tRNA molecule, reinsert the gene in a cell, and examine the in vivo charging and translation properties of the altered molecule. The ability to be charged by alanine synthetase is specified by the identity of just two nucleotides (Fig. 7.6). This was determined by identifying the smallest common subset of nucleotide changes that permitted the molecule to be charged with alanine. These proved to be two nucleotides in the acceptor stem, a G and a U that form a non Watson-Crick base pair. Providing these two nucleotides in any tRNA molecule enables the molecule to be charged with alanine. The specificity determinants of other tRNA molecules have been found to be three or more nucleotides scattered around the molecule.
The structure of the crystallized glutamyl-tRNA synthetase-tRNA complex permitted direct examination of the contacts between the enzyme and the tRNA. These showed, as expected, that this enzyme read the anticodon of the tRNA plus several nucleotides located elsewhere on the tRNA.
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