Helix 69 Is Key for Uniformity during Substrate Selection on the Ribosome

Abstract

Structural studies of ribosome complexes with bound tRNAs and release factors show considerable contacts between these factors and helix 69 (H69) of 23 S rRNA. Although biochemical and genetic studies have provided some general insights into the role of H69 in tRNA and RF selection, a detailed understanding of these contributions remains elusive. Here, we present a presteady-state kinetic analysis establishing that two distinct regions of H69 make critical contributions to substrate selection. The loop of H69 (A1913) forms contacts necessary for the efficient accommodation of a subset of natural tRNA species, whereas the base of the stem (G1922) is specifically critical for UGA codon recognition by the class 1 release factor RF2. These data define a broad and critical role for this centrally located intersubunit helix (H69) in accurate and efficient substrate recognition by the ribosome.The ribosome is the ribonucleoprotein machine responsible for the faithful translation of the genetic material into the encoded polypeptide. The core RNA components of the ribosome (the 16 S and 23 S rRNA) that reflect its earliest evolution play a key role in interactions that specify the selection of tRNAs and release factors on recognition of sense and stop codons, respectively. During translation elongation, the selection of a cognate ternary complex (composed of aa-tRNA, EFTu, and GTP) from solution is specified primarily in the small ribosomal subunit where three nucleotides in 16 S rRNA directly evaluate the geometry of the codon-anticodon interaction (1). Though different in molecular detail, the selection of class 1 release factors takes place in the same decoding center of the small ribosomal subunit where conserved protein features of the RF engage the stop codon. Thus, for both decoding events, molecular interactions in the small subunit "decoding center" are early and critical contributors to the selection of cognate substrates during the translational cycle.In addition to these interactions in the decoding center, it is believed that other molecular interactions between the ribosome and these substrates contribute to binding and specificity. Structural studies of tRNA (2-5), release factors (6 -8), and ribosome recycling factor (9, 10) bound to the ribosome reveal that H69 2 of the large subunit rRNA makes extensive contacts with all of these factors. This universally conserved helix forms an intersubunit bridge that is proximal to the substrate binding cleft and directly contacts the functionally critical h44 of 16 S rRNA which contains two of the three nucleotides known to directly interact with the decoding helix (codon-anticodon) during tRNA selection (A1492 and A1493).What is the specific nature of these molecular interactions, and what do they suggest about the role of H69 in ribosome function? H69 contacts incoming tRNA in both the pre-and postaccommodation steps near positions 25/26 of the D stem and position 38, located near the anticodon stem; tRNA mutations at these positions are linked to miscoding pheno...

Modulation of conformational changes in helix 69 mutants by pseudouridine modifications

“…These changes occur due to protonation of nucleotides that have been yet to be identified, but are likely to be conserved within the loop of H69. Furthermore, NMR spectral evidence of the loop-stem crosstalk within H69 is directly observed in pH-dependent structural changes of U/Ψ1917C, consistent with suggestions in the literature that H69 may not simply form simultaneous contacts with the A- or P-site tRNAs [38] or RFs [39, 45], but instead plays a role in coordinating the activities of the tRNAs and RFs [27–29, 42]. …”

“…Mutations at key loop residues, such as 1917, involved in mediating this crosstalk, may also be responsible for monitoring interactions with the P-site tRNA and other translational factors, e.g. , A-site tRNA or release factors (RFs), in complete ribosomes [27–29, 42], and therefore responsible for lethality in E. coli .…”

Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

“…The three pseudouridines stabilizing H69 69 by enhancing base stacking 70 are depicted in blue. The methyl group (yellow) on m 3 Y1917 in H69 prevents potential base-pairing with A1913, a residue important for uniform tRNA selection 71 . Note the flipped out conformation of A1913 facilitating interaction with the 29 OH of m 2 s 6 iA37 of the distorted Phe-tRNA Phe (purple), which, in turn, stacks onto A36 of the tRNA anticodon.…”

The Kissing-Loop T-Shaped Structure Translational Enhancer of Pea Enation Mosaic Virus Can Bind Simultaneously to Ribosomes and a 5′ Proximal Hairpin

“…Two of the three 25S rRNA helices (H69 and H89) and one of the four 18S rRNA helices (h44) exhibiting major structural changes in the presence of the kl-TSS directly contact A-site-binding tRNAs. Significant flexibility changes were found in the terminal loop of large-subunit helix H69, which participates in the B2a intersubunit bridge and makes extensive contacts with A-site tRNA and polypeptide tRNA mimics that enter the A site, such as release factors and ribosome recycling factor (55)(56)(57). H89, which lines the aminoacyl-tRNA accommodation corridor, also exhibits major flexibility changes in the presence of the kl-TSS, as does the junction region of the three helices (H90, H91, and H92) that line the other side of the accommodation corridor.…”