Abstract
The structure of the genetic code implies strict Watson–Crick base pairing in the first two codon positions, while the third position is known to be degenerate, thus allowing wobble base pairing. Recent crystal structures of near-cognate tRNAs accommodated into the ribosomal A-site, however, show canonical geometry even with first and second position mismatches. This immediately raises the question of whether these structures correspond to tautomerization of the base pairs. Further, if unusual tautomers are indeed trapped why do they not cause errors in decoding? Here, we use molecular dynamics free energy calculations of ribosomal complexes with cognate and near-cognate tRNAs to analyze the structures and energetics of G-U mismatches in the first two codon positions. We find that the enol tautomer of G is almost isoenergetic with the corresponding ketone in the first position, while it is actually more stable in the second position. Tautomerization of U, on the other hand is highly penalized. The presence of the unusual enol form of G thus explains the crystallographic observations. However, the calculations also show that this tautomer does not cause high codon reading error frequencies, as the resulting tRNA binding free energies are significantly higher than for the cognate complex.
Highlights
The ribosome ensures high speed and accuracy in translation [1,2,3,4] by selecting the correct aminoacyl-tRNA specific to the mRNA codon presented in the ribosomal A-site, from the pool of aa-tRNAs
Any deviation from standard Watson–Crick geometry at the first two codon positions resulting from near-cognate (A-C or G-U) mismatches will be strongly sensed by the monitoring bases, causing efficient rejection of the incorrect tRNA [2,4,13]
Spheres of radius 25 Acentered on the N1 atom of the first codon position were cut out from the crystal structures and solvated by a 37 Aradius water droplet, where water molecules at the sphere boundary were subjected to radial and polarization restraints according to the SCAAS model [29,31]
Summary
The ribosome ensures high speed and accuracy in translation [1,2,3,4] by selecting the correct aminoacyl-tRNA (aatRNA) specific to the mRNA codon presented in the ribosomal A-site, from the pool of aa-tRNAs. It has been suggested that the ribosome recognizes correct codon-anticodon Watson– Crick geometry by its shape and achieves high accuracy by interaction with the so-called monitoring rRNA bases of the 30 S subunit, concomitant with a small 30S interdomain movement [5]. These monitoring bases, A1492, A1493 and G530, adopt significantly different conformations in the case of an empty A-site [6,7,8] and an A-site into which a tRNA (or anticodon stem-loop) has relaxed [5,9,10,11]. TRNA modifications have a prominent role in expanding the decoding capacity and maintaining fidelity [14,15,16,17,18,19] and is has, for example, been shown that modifications at residue 34 of the tRNA anticodon can both expand and restrict the ability to recognize multiple codons [14,15,16,17,18]
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