To increase the accuracy of this binding reaction, the ribosome and EF-Tu work together in the following ways. First, the 16s rRNA in the small subunit of the ribosome assesses the “correctness” of the codon–anticodon match by folding around it and probing its molecular details (Figure 6–66). When a correct match is found, the rRNA closes tightly around the codon–anticodon pair, causing a conformational change in the ribosome that triggers GTP hydrolysis by EF-Tu. Only when GTP is hydrolyzed does EF-Tu release its grip on the aminoacyl-tRNA and allow it to be used in protein synthesis. Incorrect codon–anticodon matches do not readily trigger this conformational change, and these errant tRNAs mostly fall off the ribosome before they can be used in protein synthesis. Proofreading, however, does not end here.
After GTP is hydrolyzed and EF-Tu dissociates from the ribosome, there is a second opportunity for the ribosome to prevent an incorrect amino acid from being added to the growing chain. There is a short time delay as the amino acid carried by the tRNA moves into position on the ribosome. This time delay is shorter for correct than incorrect codon–anticodon pairs. Moreover, incorrectly matched tRNAs dissociate more rapidly than those correctly bound because their interaction with the codon is weaker. Thus, most incorrectly bound tRNA molecules (as well as a significant number of correctly bound molecules) will leave the ribosome without being used for protein synthesis. The two proofreading steps, acting in series, are largely responsible for the 99.99% accuracy of the ribosome in translating RNA into protein.
Even if the wrong amino acid slips through the proofreading steps just described and is incorporated onto the growing polypeptide chain, there is still one more opportunity for the ribosome to detect the error and provide a solution, albeit one that is not, strictly speaking, proofreading. An incorrect codonanticodon interaction in the P site of the ribosome (which would occur after the misincorporation) causes an increased rate of misreading in the A site. Successive rounds of amino acid misincorporation eventually lead to premature termination of the protein by release factors, which are described below. Normally, these release factors act when translation of a protein is complete; here, they act early. Although this mechanism does not correct the original error, it releases the flawed protein for degradation, ensuring that no additional peptide synthesis is wasted on it.
Figure 6–66 Recognition of correct codon–anticodon matches by the small-subunit rRNA of the ribosome. Shown here is the interaction between a nucleotide of the small-subunit rRNA and the first nucleotide pair of a correctly paired codon–anticodon. Similar interactions form between other nucleotides of the rRNA and the second and third positions of codon– anticodon pair. The small-subunit rRNA can form this network of hydrogen bonds only when an anticodon is correctly matched to a codon. As explained in the text, this codon–anticodon monitoring by the small-subunit rRNA increases the accuracy of protein synthesis. (From J.M. Ogle et al., Science 292:897–902, 2001. With permission from AAAS.)
Many Biological Processes Overcome the Inherent Limitations of Complementary Base-Pairing
We have seen in this and the previous chapter that DNA replication, repair, transcription, and translation all rely on complementary base-pairing—G with C, and A with T (or U). However, if only the difference in hydrogen bonding is considered, a correct versus incorrect match should differ in affinity only by a factor of 10to 100-fold. These processes have an accuracy much higher than can be accounted for by this difference. Although the mechanisms used to “squeeze out” additional specificity from complementary base-pairing differ from one process to the next, two principles exemplified by the ribosome appear to be general.