How Bacterial Ribosomal Protein L20 Assembles with 23 S Ribosomal RNA and Its Own Messenger RNA

Abstract

In bacteria, the expression of ribosomal proteins is often feedback-regulated at the translational level by the binding of the protein to its own mRNA. This is the case for L20, which binds to two distinct sites of its mRNA that both resemble its binding site on 23 S rRNA. In the present work, we report an NMR analysis of the interaction between the C-terminal domain of L20 (L20C) and both its rRNA-and mRNA-binding sites. Changes in the NMR chemical shifts of the L20C backbone nuclei were used to show that the same set of residues are modified upon addition of either the rRNA or the mRNA fragments, suggesting a mimicry at the atomic level. In addition, small angle x-ray scattering experiments, performed with the rRNA fragment, demonstrated the formation of a complex made of two RNAs and two L20C molecules. A low resolution model of this complex was then calculated using (i) the rRNA/L20C structure in the 50 S context and (ii) NMR and small angle x-ray scattering results. The formation of this complex is interesting in the context of gene regulation because it suggests that translational repression could be performed by a complex of two proteins, each interacting with the two distinct L20-binding sites within the operator.Ribosome assembly is a complex process in which proteins must assemble onto the ribosomal RNA in an ordered fashion. This process has been extensively analyzed for bacterial ribosomal subunits, using in vitro reconstitution studies. In the case of Escherichia coli large 50 S subunit, it has been shown that nine "core proteins" L1, L3, L4, L9, L10, L11, L20, L23, and L24 bind directly to the 23 S rRNA, independently of other proteins (1, 2). The other 50 S ribosomal proteins depend upon those primary binders for attachment to the large ribosomal subunit. Interestingly, several of these core proteins, namely L1, L4, L10, and L20, are also involved in the feedback regulation that allows the coordinated expression of the various ribosomal components (3-8). A similar situation is also observed for the small ribosomal subunit where regulatory ribosomal proteins are also primary 16 S rRNA binders.Most bacterial ribosomal protein genes are clustered in polycistronic operons, the expression of which is controlled at the translational level (9 -11). One cistron encodes a regulatory ribosomal protein that binds to the messenger RNA, thereby shutting off the translation of the downstream ribosomal protein cistrons. In the classical Nomura model, it is assumed that the protein-binding site on the mRNA, the translational operator, is structurally similar to its binding site on the rRNA. Molecular mimicry between the two target RNA sites would then be used to adjust the translation of ribosomal proteins to the level of transcription of the rRNA; in the presence of excess unbound rRNA, the repressor ribosomal protein would be displaced from its mRNA, thereby allowing translation to proceed. So far, in E. coli, the molecular mimicry between the translational operator on the mRNA and the rRNA has been demonstrate...

Escherichia coli ribosomal protein L20 binds as a single monomer to its own mRNA bearing two potential binding sites

“…The 1:1 stoichiometry demonstrated here for the L20/operator complex was somewhat surprising in light of our earlier structural studies of the interaction of L20 with RNA (14). In these studies, the complex between the C-terminal fragment of L20 and a 42-nt long oligoribonucleotide derived from the rRNA-binding site for L20 was modelled using a combination of structural data from the ribosome, NMR and SAXS.…”

“…The eluted peaks were monitored at 280 nm. Escherichia coli L20C-ter was prepared and purified essentially as described for the C-terminal domain of Aquifex æolicus L20 (14). …”

“…Also, NMR footprinting experiments indicate that the same region of L20 is responsible for the interaction with the two mRNA and the rRNA sites, i.e. L20 has only one RNA-binding site allowing the recognition of the three RNA structures (12,14). The presence of these two binding sites on the mRNA and only one binding site on the protein suggested either that L20 recognizes its mRNA as a dimer or that two monomeric L20 molecules bind independently to each of the mRNA sites.…”

Recombinant RNA technology: the tRNA scaffold

“…By immobilizing RNA chimeras on the affinity matrix, it is possible to perform specific pull down assays, e.g. the specific binding of ribosomal protein L20 to its target site on the 23S rRNA 27 (Fig. 4c).…”

The NMR Structure of an Internal Loop from 23S Ribosomal RNA Differs from Its Structure in Crystals of 50S Ribosomal Subunits,

“…L20 is one of nine core proteins (13,14) that bind to 23S rRNA during an early stage of ribosomal assembly in E. coli (15). The C-terminal domain of L20 represses the translation of its own mRNA and it has been suggested that its binding site on mRNA mimics the binding site on the rRNA (10,16). The residues at the interface of the protein with helix 40 of 23S rRNA are conserved and involved in recognition of rRNA (17).…”