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.
Highlights
Single molecule fluorescence resonance energy transfer, crystallographic and cryo-electron microscopy techniques have been used to obtain high resolution structures of ribosomes in various stages of its motions during translation as well as its various components (Frank and Agrawal 2000; Valle et al 2003; Gagnon et al 2016; Shebl et al 2016; Wasserman et al 2016; Zhou et al 2020)
In order to address this issue, cryo-electron microscopy (cryo-EM) was used to characterize the 3D structure of the native large subunit of H. morrhuae (ATCC® 10782)
We purified large ribosomal subunits from H. morrhuae ATCC® 17082 and imaged them by cryo-EM. This allowed us to obtain a 3D model of the 5S rRNA in the context of the intact large subunit
Summary
Single molecule fluorescence resonance energy transfer (smFRET), crystallographic and cryo-electron microscopy (cryo-EM) techniques have been used to obtain high resolution structures of ribosomes in various stages of its motions during translation as well as its various components (Frank and Agrawal 2000; Valle et al 2003; Gagnon et al 2016; Shebl et al 2016; Wasserman et al 2016; Zhou et al 2020). Insertions typically are accommodated in the ribosome by forming a three or four-way junction with negligible perturbation of the parental helix (Petrov et al 2014). 17082), which contains a large 108 nucleotide insertion (Luehrsen et al 1981) that almost doubles the size of the RNA (Fig. 1).
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