Pseudouridine-Free Escherichia coli Ribosomes

Abstract

Pseudouridine (Ψ) is present at conserved, functionally important regions in the ribosomal RNAs (rRNAs) from all three domains of life. Little, however, is known about the functions of Ψ modifications in bacterial ribosomes. An strain has been constructed in which all seven rRNA Ψ synthases have been inactivated and whose ribosomes are devoid of all Ψs. Surprisingly, this strain displays only minor defects in ribosome biogenesis and function, and cell growth is only modestly affected. This is in contrast to a strong requirement for Ψ in eukaryotic ribosomes and suggests divergent roles for rRNA Ψ modifications in these two domains. Pseudouridine (Ψ) is the most abundant posttranscriptional modification in RNAs. In the ribosome, Ψ modifications are typically located at conserved, critical regions, suggesting they play an important functional role. In eukarya and archaea, rRNAs are modified by a single pseudouridine synthase (PUS) enzyme, targeted to rRNA via a snoRNA-dependent mechanism, while bacteria use multiple stand-alone PUS enzymes. Disruption of Ψ modification of rRNA in eukarya seriously impairs ribosome function and cell growth. We have constructed an multiple deletion strain lacking all Ψ modifications in rRNA. In contrast to the equivalent eukaryotic mutants, the strain is only modestly affected in growth, decoding, and ribosome biogenesis, indicating a differential requirement for Ψ modifications in these two domains.

Mining for Perchlorate Resistance Genes in Microorganisms From Sediments of a Hypersaline Pond in Atacama Desert, Chile

“…This enzyme modifies uridine into pseudouridine at certain positions of the 23S rRNA and tRNAs (Raychaudhuri et al, 1999). It was suggested that these modifications play a role in the stability of RNA and ribosome biogenesis (Cabello-Villegas and Nikonowicz, 2005;O'Connor et al, 2017). Furthermore, RNase E participates in rRNA and tRNA maturing processes (Mackie, 2013), and therefore it could increase the levels of these RNA molecules.…”

Translational adaptation to heat stress is mediated by RNA 5‐methylcytosine in Caenorhabditis elegans

“…Chemical modifications of RNA occur in organisms from all kingdoms of life and are often highly conserved throughout evolution, as is the case of the methylation of carbon-5 in cytosines (Huber et al, 2015;Boccaletto et al, 2017). Despite that, there is a growing body of evidence showing that several RNA modifications are individually not required for development under controlled conditions (O'Connor et al, 2018, reviewed in Sharma & Lafontaine, 2015and Hopper & Phizicky, 2003. Our results reignite a recurrent question in the epitranscriptomics field: why are so many of these chemical marks extensively conserved throughout evolution and, yet, organisms often present subtle phenotypes in their absence?…”

Cryo-electron microscopy structure of the 70S ribosome from Enterococcus faecalis

“…These include single nucleotide substitutions and deletion of a nucleotide in the tip of H69 43 . Furthermore, removal of three pseudo-uridylation sites (PSU 1911, 1915, and 1917) by deletion of the responsible pseudo-U synthase RluD causes little to no phenotype in E. coli 44 . While complete deletion of H69 does not affect translation significantly, ΔH69 50S subunits show subunit association defects, emphasizing the role of this bridge for stabilizing 70S ribosomes 45 .…”