Principles of stop-codon reading on the ribosome

Abstract

In termination of protein synthesis, the bacterial release factors RF1 and RF2 bind to the ribosome through specific recognition of messenger RNA stop codons and trigger hydrolysis of the bond between the nascent polypeptide and the transfer RNA at the peptidyl-tRNA site, thereby releasing the newly synthesized protein. The release factors are highly specific for a U in the first stop-codon position and recognize different combinations of purines in the second and third positions, with RF1 reading UAA and UAG and RF2 reading UAA and UGA. With recently determined crystal structures of termination complexes, it has become possible to decipher the energetics of stop-codon reading by computational analysis and to clarify the origin of the high release-factor binding accuracy. Here we report molecular dynamics free-energy calculations on different cognate and non-cognate termination complexes. The simulations quantitatively explain the basic principles of decoding in all three codon positions and reveal the key elements responsible for specificity of the release factors. The overall reading mechanism involves hitherto unidentified interactions and recognition switches that cannot be described in terms of a tripeptide anticodon model. Further simulations of complexes with tRNA(Trp), the tRNA recognizing the triplet codon for Trp, explain the observation of a 'leaky' stop codon and highlight the fundamentally different third position reading by RF2, which leads to a high stop-codon specificity with strong discrimination against the Trp codon. The simulations clearly illustrate the versatility of codon reading by protein, which goes far beyond tRNA mimicry.

Codon-reading specificities of mitochondrial release factors and translation termination at non-standard stop codons

“…3a (see also Supplementary Table S1), and provide a clear picture of the recognition pattern of the two mitochondrial RFs. As found earlier 20 , the calculated binding free energies for RF1 are very similar to those derived from experimental K M values 21 . Both mtRF1a and mtRF1 are predicted to behave qualitatively the same as RF1, with large discriminations towards both a firstposition A and a second-position G. This is evident from both mutations of UAA into AAA and UGA, and the mutation of UGA into AGA.…”

“…Hence, it is evident for both mtRF1a and mtRF1 that there is too limited space for accommodating a purine at the first stop codon position owing to the location of the a5 helix. In particular, none of the mtRF1 insertion residues show any sign of favourable interactions with a first-position A, in contrast to the UAA codon, which shows the same strong hydrogen bonds with the a5 helix and the recognition loop that have previously been identified 14,20 .…”

“…Just as for RF1, both mtRF1a and mtRF1 are found to be insensitive to A/G substitution at the third codon position, in accordance with the conservation of the key RF1 residues Gln181 and Thr194 (ref. 20).…”

“…The key residue enabling reading of a second-position G in RF2 is Glu128, the position of which is controlled by specific interaction network of mostly charged residues 20,23 . Although this residue is also a glutamate in RF1 (Glu119) and mtRF1a (Glu141), its interaction network is different in these RFs, prohibiting an efficient interaction with G2 as described earlier 20 .…”

“…Neither of these hypotheses can be completely verified or disproved without an in vitro vertebrate mitochondrial translation assay, but no such system is available at present. However, computational modelling and molecular dynamics (MD) simulations have been remarkably successful in predicting key features of several steps involved in the translation process 18 , including termination by class-1 RFs 19,20 , and may offer a useful strategy for exploring the mitochondrial termination problem.…”