Double-sieving

The suppressor tRNA developed by the Chamberlin lab for use in a rabbit reticulocyte lysate is based on an E. coli glycyl tRNA, which was initially chosen because glycyl-tRNA synthetases do not rely on a double-sieve editing mechanism for enzymatic hydrolysis of misacylated tRNAs [26]. Two base pair changes were made to the acceptor stem to allow incorporation of the optimal T7 RNA polymerase promoter into the DNA template for tRNA y-Con [27,28],... 

Figure 13.4 The double sieve analogy for the editing mechanism of the isoleucyl-tRNA synthetase. The active site for the formation of the aminoacyl adenylate can exclude amino acids that are larger than isoleucine but not those that are smaller. On the other hand, a hydrolytic site that is just large enough to bind valine can exclude isoleucine while accepting valine and all the smaller amino acids. (In some enzymes, the hydrolytic site offers specific chemical interactions that enable it to bind isosteres of the correct amino acid as well as smaller amino acids.)...

In this case, / = / /". For the isoleucyl-tRNA synthetase, / 150, so any subsequent increase in specificity is limited to a factor of less than 15 if the cost is to be kept tolerable. In fact, the measured cost is less than 0.05, and it may be predicted from the cost-selectivity equation that if the Hopfield mechanism were operating it would be increasing specificity by a factor of less than 8.5.55 We show next that double sieving is not limited in the same way. 

Double mutant cycles—see thermodynamic cycles Double-sieve editing mechanisms 387-389, 398, 399... 

Fukai, S., Nureki, O., Sekine, S., Shimada, A., Tao,J., Vassylyev, D. G., and Yokoyama, S. (2000). Structural basis for double-sieve discrimination of L-valine from L-isoleucine and L-threonine by the complex of tRNA(Val) and valyl-tRNA synthetase. Cell 103, 793-803. 

Most aaRSs that edit use a double-sieve mechanism. The aminoacylation active site serves as a coarse sieve and binds cognate amino acid as well as structurally related noncognate amino acids. A second hydrolytic active site acts as a fine sieve and hydrolyzes misactivated amino acids. The Class 1... 

Most aminoacyl-tRNA synthetases contain editing sites in addition to acylation sites. These complementary pairs of sites fimction as a double sieve to ensure very high fidelity. In general, the acylation site rejects amino acids that are larger than the correct one because there is insufficient room for them, whereas the hydrolytic site cleaves activated species that are smaller than the correct one. 

---