In-cell SHAPE reveals that free 30S ribosome subunits are in the inactive state

Abstract

It was shown decades ago that purified 30S ribosome subunits readily interconvert between "active" and "inactive" conformations in a switch that involves changes in the functionally important neck and decoding regions. However, the physiological significance of this conformational change had remained unknown. In exponentially growing Escherichia coli cells, RNA SHAPE probing revealed that 16S rRNA largely adopts the inactive conformation in stably assembled, mature 30S subunits and the active conformation in translating (70S) ribosomes. Inactive 30S subunits bind mRNA as efficiently as active subunits but initiate translation more slowly. Mutations that inhibited interconversion between states compromised translation in vivo. Binding by the small antibiotic paromomycin induced the inactiveto-active conversion, consistent with a low-energy barrier between the two states. Despite the small energetic barrier between states, but consistent with slow translation initiation and a functional role in vivo, interconversion involved large-scale changes in structure in the neck region that likely propagate across the 30S body via helix 44. These findings suggest the inactive state is a biologically relevant alternate conformation that regulates ribosome function as a conformational switch. orty-five years ago, Zamir, Elson, and their colleagues reported that purified 30S subunits of the ribosome undergo a readily reversible conformational change between "active" and "inactive" states and proposed that this conformational rearrangement might mimic a natural process (1). Noller and coworkers used chemical probing to show that this conformational change occurs in the neck and decoding center regions of the 16S ribosomal RNA (rRNA) and has "the appearance of a reciprocal interconversion between two differently structured states" (2). Recent structural analyses indicate that the protein-free 16S rRNA adopts alternative base-paired conformations in the neck region that are conserved among diverse eubacterial and archeal organisms (3). The ability to sample multiple conformations in this region is also conserved in eukaryotes (4). The original studies on the inactive and active states noted that probing ribosomes in cells might allow the biological roles of these states to be established (1, 2). Here we make use of recent innovations in in-cell RNA SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) probing (5) to interrogate the structure of 16S rRNA in free 30S subunits, in actively translating ribosomes, and in mutant ribosomes in exponentially growing Escherichia coli. ResultsIn Vivo SHAPE Probing of Ribosomal States. We used in vivo SHAPE (5, 6) to probe the RNA structure in exponentially growing E. coli cells and then halted translation by rapidly pouring the cells over ice (7). Experiments were performed with the SHAPE reagent 1M7, which readily enters cells and either reacts with RNA or undergoes inactivation by hydrolysis over ∼2 min. Probing is thus rapid, no explicit quench step is required, and the ex...

Comparison of SHAPE reagents for mapping RNA structures inside living cells

“…However, recent reports have suggested that 1M7, an isatoic anhydride reagent related to NMIA, was able to robustly modify both ribosomal RNA as well as mRNAs inside both mammalian and bacterial cells (McGinnis et al 2015;Smola et al 2015;Takahashi et al 2016;Watters et al 2016). These apparently contradictory results stimulated us to further investigate the differences between the two types of SHAPE reagents.…”

“…Published work has reported in vivo modification of RNA in E. coli cells; however, no cDNA truncation blots were shown (McGinnis et al 2015;Smola et al 2015;Takahashi et al 2016). To address the possibility that our lack of in vivo signal using 1M7 is due to the type of cells used, we repeated the SHAPE experiment in HST08 E. coli cells following precisely the published protocols for cell culture, RNA modification, and RNA extraction (McGinnis et al 2015).…”

“…To address the possibility that our lack of in vivo signal using 1M7 is due to the type of cells used, we repeated the SHAPE experiment in HST08 E. coli cells following precisely the published protocols for cell culture, RNA modification, and RNA extraction (McGinnis et al 2015). As with the mouse samples, all four acylation reagents were successful at measuring RNA structure stops from in vitro modified RNA; however, only 1M7 was not able to generate robust data from live cell modification (Fig.…”

Conserved GTPase LepA (Elongation Factor 4) functions in biogenesis of the 30S subunit of the 70S ribosome

“…1). The large 70S peak of these gradients includes translating monosomes as well as ribosomes that finish translation before cell lysis (8)(9)(10). Bearing this in mind, we infer that SSU particles lacking S3, S10, S14, and S21 accumulate in the ΔlepA mutant but do not efficiently enter the actively translating pool of ribosomes.…”

Data-directed RNA secondary structure prediction using probabilistic modeling

“…Taking this notion a step further, if we generated improved models of the data and concurrently integrated them into model-based decoding schemes such as RNAprob, then this has the potential to lead to better predictions. Recently, McGinnis et al (2015) showed that free 30S ribosome subunits are in the inactive state. In particular, the functionally important h28-h44 region contains an alternative structure inconsistent with the conventional reference.…”