Translation proofreading

The functional roles of rRNAs include the participation in mRNA selection, tRNA binding, ribosomal subunit association, frame-shift snppression, translational proofreading, binding of various factors and peptidyl transferase activity. Messenger RNA is selected by interaction with the 3 -terminus of 16S rRNA in the platform region of the small subunit. tRNA interacts with 16S rRNA in the cleft of the small subunit and around the universally conserved central loop of 23 S rRNA (via CCA terminal of tRNA) of the large subunit. This conserved region of rRNA may be involved in the peptidyl transferase activity. Subunit interaction appears to involve elements of both 16S and 23S rRNA. 

In this way the erroneous incorporation of valine in place of isoleucine is avoided. The sequential action of aminoacylation and proofreading sites contributes to an overall error frequency of translation of less than 1 in 10,000. 

Other factors, too numerous to mention in this brief sketch, are also necessary for a functioning translation system. These include the enzymes that chemically place the correct amino acid onto the correct tRNA, various mechanisms to proofread the translation, and the role of chemical energy, in the form of the activated nucleotide GTP at every stage of translation. Nonetheless, this outline may give the reader both an idea of the process by which genetic information is expressed and also an appreciation for the intricacies involved in that expression. 

I would like to thank Eric Bett, Elisabeth Gotschi, Pamela Pali and Frank Place for comments and proofreading, Richard Onwonga for detailed discussions and contributions on Rift Valley and Tena Mimica for translation support. 

Blanchard SC, Gonzalez RL, Kim HD, Chu S, Puglisi JD. tRNA selection and kinetic proofreading in translation. Nat. Struct. Mol. Biol. 2004 11 1008-1014. 

Without the help and understanding of my husband Dr. Stefan Brammer this book would have never come into existence. I thank him for the never-ending support, motivation, proofreading, translating, and everything else which helped to finish this project. 

Because some amino acids are so similar structurally, aminoacyl-tRNA synthetases sometimes make mistakes. These are corrected, however, by the enzymes themselves, which have a proofreading activity that checks the fit In their amino acid-binding pocket. If the wrong amino acid becomes attached to a tRNA, the bound synthetase catalyzes removal of the amino acid from the tRNA. This crucial function helps guarantee that a tRNA delivers the correct amino acid to the protein-synthesizing machinery. The overall error rate for translation In E. coli Is very low, approximately 1 per... 

Aminoacyl tRNA-synthetases have a proofreading ability to double-check that an amino acid is linked with its proper tRNA. The proofreading ability of the enzyme and other proofreading steps (see here) in translation reduce the error frequency to less than 1 in 10,000. 

The recognition of the correct tRNA by the synthetase is vital to the fidelity of translation because most of the final proofreading occurs at this step. See the articles by LaRiviere et al. and Ibba cited in the bibliography at the end of this chapter for the latest information on this topic. 

Taq polymerase has no 3 -proofreading exonuclease activity. Thus it can misincor-porate bases. However, the enzyme does have a 5 -exonuclease activity and therefore will nick translate. The most serious problem generally encountered is that Taq polymerase can add an extra nontanplate-coded A to the 3 -end of DNA chains. It can also use a related activity, making a primer dimeric that may or may not contain additional uncoded residues. Once such dimeric primers are created, they are efficient substrates for further amplification. These primer dimers are a major source of artifacts in PCR. However, they are usually... 

---