Cryo-electron microscopy visualization of a large insertion in the 5S ribosomal RNA of the extremely halophilic archaeon Halococcus morrhuae - PubMed (original) (raw)

Cryo-electron microscopy visualization of a large insertion in the 5S ribosomal RNA of the extremely halophilic archaeon Halococcus morrhuae

Madhan R Tirumalai et al. FEBS Open Bio. 2020 Oct.

Abstract

The extreme halophile Halococcus morrhuae (ATCC® 17082) contains a 108-nucleotide insertion in its 5S rRNA. Large rRNA expansions in Archaea are rare. This one almost doubles the length of the 5S rRNA. In order to understand how such an insertion is accommodated in the ribosome, we obtained a cryo-electron microscopy reconstruction of the native large subunit at subnanometer resolution. The insertion site forms a four-way junction that fully preserves the canonical 5S rRNA structure. Moving away from the junction site, the inserted region is conformationally flexible and does not pack tightly against the large subunit. The high-salt requirement of the H. morrhuae ribosomes for their stability conflicted with the low-salt threshold for cryo-electron microscopy procedures. Despite this obstacle, this is the first cryo-electron microscopy map of Halococcus ribosomes.

Keywords: accretion model; archaea; expansion sequences; insertion sequences; ribosomal RNA.

© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1

Fig. 1

An unusual 5S rRNA insertion. A schematic diagram showing (A) a possible secondary structure of the insert as predicted by mfold [50] and RNAstructure [51] and (B) the usual 5S rRNA secondary structure model (available at RNAcentral (

https://rnacentral.org/

) and the Comparative RNA Web (CRW) Site(

http://www.rna.ccbb.utexas.edu

)) [52, 53] and modified; the insert is between positions U104 and G105 as per Halococcus morrhuae 5S rRNA numbering. The equivalent positions in Haloarcula marismortui are C108 and G109. What would be the 5′ and 3′ ends of the insert if it were an independent RNA are indicated in (A).

Fig. 2

Fig. 2

Cryo‐EM reconstruction of the large subunit of Halococcus morrhuae. (A) The large subunit of Haloarcula marismotui (PDB:

1NJI

) [49] is rigidly docked in the density obtained from 3D cryo‐EM reconstruction; 5S rRNA (purple), 23S rRNA (yellow), and uL5 and uL18 (orange‐red), and selected ribosomal other proteins (cyan) are shown; the insert is marked by *; Cp—central protuberance. The scale bar indicates 50 Å. (B) A view of the large subunit with the best‐available crystal structure docked in (PDB:

4V9F

) [48] rotated to focus on the discordance in the helix 25 path (red arrow summarizing the crystal structure and green arrow summarizing the cryo‐EM structure). The cryo‐EM density at lower isosurface threshold is shown as an outline around the map, illustrating the continued projection of the helix. The P stalk can be seen on the left edge; though the P stalk proteins L11, L12, and L10e are visualized in the crystal structure of Haloarcula marismotui, they are not seen in this cryo‐EM map. Scale is identical to panel A. (C) Plot of the consistency of the two half‐maps after tight masking and mask correction. The 0.143 ‘gold‐standard’ cutoff is indicated as a dashed line.

Fig. 3

Fig. 3

3D structure of the 5S rRNA. (A) The large subunit of Haloarcula marismotui (PDB:

1NJI

) [49] is docked in the density (left, view from the 30S side; right, view from the solvent side of the 50S subunit); only the 5S rRNA is shown colored, and the insert is marked by *. Coloring is by nucleotide: adenines, green; cytosines, orange; guanines, red; and uracils, blue. Cp—central protuberance; St—stalk. The scale bar indicates 50 Å. (B) The section of the map corresponding to the 5S rRNA is cropped out for visualization. The Haloarcula marismortui 5S rRNA was mutated to match the sequence of the Halococcus morrhuae 5S, except that the insert (whose secondary structure is not known) was omitted, and rigidly docked inside the density. The insertion site is marked by *. The scale bar indicates 5 Å. (C) The section of the map corresponding to the 5S rRNA is cropped out of the published, 6.6 Å Methanothermobacter thermautotrophicus cryo‐EM map and associated atomic model (EMD‐2012, PDB

4ADX

) [37] and colored as in (B). This and all other homolog maps lack the protruding lump of density seen in (B). Scale is identical to panel B.

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