Analysis of r-protein and RNA conformation of 30S subunit intermediates in bacteria

Abstract

The ribosome is a large macromolecular complex that must be assembled efficiently and accurately for the viability of all organisms. In bacteria, this process must be robust and tunable to support life in diverse conditions from the ice of arctic glaciers to thermal hot springs. Assembly of the Small ribosomal SUbunit (SSU) of Escherichia coli has been extensively studied and is highly temperature-dependent. However, a lack of data on SSU assembly for other bacteria is problematic given the importance of the ribosome in bacterial physiology. To broaden the understanding of how optimal growth temperature may affect SSU assembly, in vitro SSU assembly of two thermophilic bacteria, Geobacillus kaustophilus and Thermus thermophilus, was compared with that of E. coli. Using these phylogenetically, morphologically, and environmentally diverse bacteria, we show that SSU assembly is highly temperature-dependent and efficient SSU assembly occurs at different temperatures for each organism. Surprisingly, the assembly landscape is characterized by at least two distinct intermediate populations in the organisms tested. This novel, second intermediate, is formed in the presence of the full complement of r-proteins, unlike the previously observed RI * particle formed in the absence of late-binding r-proteins in E. coli. This work reveals multiple distinct intermediate populations are present during SSU assembly in vitro for several bacteria, yielding insights into RNP formation and possible antimicrobial development toward this common SSU target.

Purification and Characterization of Authentic 30S Ribosomal Precursors Induced by Heat Shock

“…This suggests that (under our purification conditions) bS1 is not released by the 30S subunits, and that the previously observed absence of bS1 in 21S could be due to it not being incorporated at an early stage of the small subunit biogenesis. uS3 is also under-represented, as observed in most published reports on 30S precursors [ 9 , 12 , 16 , 17 , 30 ]. In fact, almost all of the proteins found in low abundance are secondary (S9, S13, and S19) or tertiary (S10, S14, S3, and S2) binding proteins that bind to the 21S complex later in the 30S maturation process.…”

The N-terminal extension of yeast ribosomal protein L8 is involved in two major remodeling events during late nuclear stages of 60S ribosomal subunit assembly

“…When an r-protein is depleted, only the earliest defect in assembly can be detected. Yet, it has been suggested that r-proteins establish several different stepwise interactions with rRNA during the assembly pathway, and thus may be involved in multiple stages of pre-RNP remodeling and pre-rRNA processing (Adilakshmi et al 2008;Kim et al 2014;Napper and Culver 2015). According to this model, the initial binding of an r-protein to rRNA forms an "encounter complex," which is then converted to more stable complexes by establishing greater numbers of contacts with rRNA.…”