Modulation of conformational changes in helix 69 mutants by pseudouridine modifications

Abstract

Centrally located at the ribosomal subunit interface and mRNA tunnel, helix 69 (H69) from 23S rRNA participates in key steps of translation. Ribosome activity is influenced by three pseudouridine modifications, which modulate the structure and conformational behavior of H69. To understand how H69 is affected by the presence of pseudouridine in combination with sequence changes, the biophysical properties of wild-type H69 and representative mutants (A1912G, U1917C, and A1919G) were examined. Results from NMR and circular dichroism spectroscopy indicate that pH-dependent structural changes of wild-type H69 and the chosen mutants are modulated by pseudouridine and loop sequence. The effects of the mutations on global stability of H69 are negligible; however, pseudouridine stabilizes H69 at low pH conditions. Alterations to induced conformational changes of H69 likely result in compromised function, as indicated by previous biological studies.

Keywords: CD; NMR; RNA; Ribosome; Thermodynamics.

Fig. 1

The location of H69 within the 70S bacterial ribosome. The figure was prepared from the PDB IDs: 3I8F and 3I8G using PyMol [4]. As part of the large ribosomal subunit, H69 is located at the interface between the large and small subunits, and positioned between the A-site and P-site tRNAs. The tRNAs, together with the mRNA, move from right to left during the translation process, as illustrated in the figure.

Fig. 2

Secondary structure of H69 and chemical structures of uridine (U) and pseudouridine (Ψ). a) Pseudouridine (Ψ) modifications at positions 1911, 1915, and 1917 (E. coli numbering) are labeled. The mutations employed in this project (i.e., A1912G, U/Ψ1917C, and A1919G) are indicated with arrows, and the complete RNA sequences are listed in the Supplementary Information, Table S1. b) Due to replacement of the N1-C1′ glycosidic bond by a C5-C1′ linkage, the N1-position in Ψ is protonated (indicated by an arrow) and available for hydrogen-bonding interactions.

Fig. 3

CD and NMR spectra of the unmodified and modified H69 A1912G mutants. The CD spectra of UUU- and ΨΨΨ-A1912G at pH 7.0 (solid line) and pH 5.5 (dashed line) are given in panels a and b, respectively. The NMR spectra of unmodified (UUU, c and d) and modified (ΨΨΨ, e and f) A1912G were obtained at pH 7.0 (c and e) or pH 5.5 (d and f) as indicated.

Fig. 4

CD and NMR spectra of the unmodified and modified H69 A1919G mutants. The CD spectra of UUU- and ΨΨΨ-A1919G at pH 7.0 (solid line) and pH 5.5 (dashed line) are given in panels a and b, respectively. The NMR spectra of unmodified (UUU, c and d) and modified (ΨΨΨ, e and f) A1919G were obtained at pH 7.0 (c and e) or pH 5.5 (d and f) as indicated.

Fig. 5

CD and NMR spectra of the unmodified and modified H69 U/Ψ1917C mutants. The CD spectra of UUC- and ΨΨC-U/Ψ1917C at pH 7.0 (solid line) and pH 5.5 (dashed line) are given in panels a and b, respectively. The NMR spectra of unmodified (UUC, c and d) and modified (ΨΨC, e and f) U/Ψ1917C were obtained at pH 7.0 (c and e) or pH 5.5 (d and f) as indicated.