The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control

Abstract

The Escherichia coli autoregulatory ribosomal protein L20 consists of two structurally distinct domains. The C-terminal domain is globular and sits on the surface of the large ribosomal subunit whereas the N-terminal domain has an extended shape and penetrates deep into the RNA-rich core of the subunit. Many other ribosomal proteins have analogous internal or terminal extensions. However, the biological functions of these extended domains remain obscure. Here we show that the N-terminal tail of L20 is important for ribosome assembly in vivo. Indeed, a truncated version of L20 without its N-terminal tail is unable to complement the deletion of rplT, the gene encoding L20. In addition, this L20 truncation confers a lethal-dominant phenotype, suggesting that the N-terminal domain is essential for cell growth because it could be required for ribosome assembly. Supporting this hypothesis, partial deletions of the N-terminal tail of the protein are shown to cause a slow-growth phenotype due to altered ribosome assembly in vivo as large amounts of intermediate 40S ribosomal particles accumulate. In addition to being a ribosomal protein, L20 also acts as an autogenous repressor. Using L20 truncations, we also show that the N-terminal tail of L20 is dispensable for autogenous control.

Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly

“…To date, a limited number of r-protein extensions have been shown to be important in bacterial ribosome assembly: those present in S4 and S12 in the 30S subunit (Mayerle and Woodson 2013;Calidas et al 2014), and the extension of L20 in the 50S subunit (Guillier et al 2005). Similarly, only a handful of mutant alleles have been identified demonstrating that the conserved extended domains of eukaryotic r-proteins are important for ribosomal subunit formation (Jakovljevic et al 2004;Bussiere et al 2012;Garcia-Gomez et al 2014).…”

The N-terminal extension of S12 influences small ribosomal subunit assembly in Escherichia coli

“…One hypothesis is that r-proteins with long extensions represent a subset of intrinsically disordered proteins whose function depends upon a structural transition from a disordered form to an ordered structure upon the binding of its partner (Uversky et al 2000;Iakoucheva et al 2002;Timsit et al 2009). For example, the N-terminal extension of r-protein L20 undergoes a helix-coil transition upon binding 23S rRNA, and is essential for large subunit assembly (Guillier et al 2005;Timsit et al 2006). Similar data for S4 in the in vitro assembly of the 30S subunit also lend credence to the idea that r-proteins with long noncanonically structured regions play an important role in assembly by cofolding with 16S rRNA (Mayerle and Woodson 2013).…”

“…However, the exact role of the extensions in bacterial ribosome function and assembly is unclear. Of the r-proteins with extensions that have been studied, complete truncation of the extension of E. coli L20 has the most profound effect, leading to a dominant lethal phenotype, while partial deletions resulted in slow growth due to defects in large subunit (LSU or 50S) assembly (Guillier et al 2005). In contrast, deletion of the long loops of E. coli L4 and L22 had no effect on the assembly of the 50S subunit (Zengel et al 2003).…”

Mutations in Residues Involved in Zinc Binding in the Catalytic Site of Escherichia coli Threonyl-tRNA Synthetase Confer a Dominant Lethal Phenotype

“…Ribosomal protein L20 is composed of two domains, a globular domain that recognizes the helix 40-helix 41 junction of 23S rRNA and a long ␣-helical N-terminal extension that contacts several quite distant ribosomal rRNA sites in domains I and II of the 23S (27). We have isolated a deletion in the gene for ribosomal protein L20 that yields a protein without its ␣-helical N-terminal extension (9). If cloned on a multicopy plasmid under control of the lac regulatory regions, this mutant allele gives a dominant lethal phenotype under induction conditions.…”