Nucleotide modifications and tRNA anticodon-mRNA codon interactions on the ribosome

Abstract

We have carried out molecular dynamics simulations of the tRNA anticodon and mRNA codon, inside the ribosome, to study the effect of the common tRNA modifications cmo(5)U34 and m(6)A37. In tRNA(Val), these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. Previous data suggest that entropic effects are mainly responsible for the extended reading capabilities, but detailed mechanisms have remained unknown. We have performed a wide range of simulations to elucidate the details of these mechanisms at the atomic level and quantify their effects: extensive free energy perturbation coupled with umbrella sampling, entropy calculations of tRNA (free and bound to the ribosome), and thorough structural analysis of the ribosomal decoding center. No prestructuring effect on the tRNA anticodon stem-loop from the two modifications could be observed, but we identified two mechanisms that may contribute to the expanded decoding capability by the modifications: The further reach of the cmo(5)U34 allows an alternative outer conformation to be formed for the noncognate base pairs, and the modification results in increased contacts between tRNA, mRNA, and the ribosome.

FIGURE 1.

The two prokaryotic ribosomal subunits, 5S (pale pink) and 23S (pale blue) together with three tRNAs bound to the A-, P-, and E-sites. The studied system is colored red.

FIGURE 2.

The ribosomal decoding center. tRNA^Val ASL in green, mRNA valine codon in blue, and ribosomal RNA in magenta. The wobble base pair and surrounding residues, participating in hydrogen bonds, are highlighted in sticks. The three codon-anticodon base pairs are numbered and specified with dashed bonds.

FIGURE 3.

(Left) N⁶-methyladenosine (m⁶A) and (right) uridine 5-oxyacetic acid (cmo⁵U).

FIGURE 4.

Root mean square deviations of all solute atoms (modified systems, seven replica simulations) for the ASL bound to cytosine (A), adenine (B), uracil (C), and guanine (D) at the third codon position. The insets show the average of the first 10 nsec for modified (black) and unmodified (red) systems.

FIGURE 5.

Quasiharmonic entropy as a function of time for the ASL free in solution (modified in black, unmodified in red).

FIGURE 6.

Relative free energy between the inner and outer (insets) conformations obtained by PMF calculations. The standard mismatch (inner) conformation has been chosen as the reference state. The reaction coordinate is the distance between O4 of cmo⁵U34 and H1, H42, and H3 for guanine (red), cytosine (black), and uracil (blue), respectively. Error bars are standard errors calculated by comparing three parts of the trajectories.

FIGURE 7.

Hydrogen bond interactions between some selected residues around the wobble base pair for the ASL (modified in blue, unmodified in green) bound to cytosine (A), adenine (B), uracil (C), and guanine (D). Direct interactions are shown in dark colors and the water-bridged are added on top with light colors. (R) Ribosomal residues, (m) mRNA, (t) tRNA.

FIGURE 8.

Observed interactions of cmo⁵U34. (A) A network of hydrogen bonds between G3 and U34 is possible with the cmo⁵-modification. (B) cmo⁵ extends the reach to help binding with U3. (C) When free in solution, the cmo⁵-modification forms several water-bridged contacts to the backbone. (D) The carboxylic oxygens of cmo⁵ bridge the gap between the ribosomal C1054 and A4 of mRNA.