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

Frédéric Allemand,Patrice Vachette,Frédéric Dardel,Maude Guillier,Claude Chiaruttini,Sophie Raibaud

doi:10.1074/jbc.m304717200

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.

Highlights

Ribosome assembly is a complex process in which proteins must assemble onto the ribosomal RNA in an ordered fashion
In order to investigate the recognition of rpmI translational operator, we decided to focus on the C-terminal domain of the protein, L20C, which corresponds to the second half of the sequence, starting approximately at the single tryptophan residue in the middle of the protein
Interaction studies were not conducted with the full-length L20 for the following reasons. (i) The C-terminal domain suffices to repress rpmI expression [23]. (ii) In the crystal structure of D. radiodurans 50 S subunit [24], the C-terminal domain of L20 interacts tightly with a discrete region of the 23 S rRNA, at the junction between helices 40 and 41 [25], which exhibits similarities with the rpmI translational operator [22]. (iii) It was suspected that the strongly basic and unfolded N-terminal domain would make spurious electrostatic contacts with the RNA fragments, yielding to nonspecific aggregation of the sample, a phenomenon observed in preliminary experiments

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The plasmid pET42aL20aa⌬N was constructed as follow: pBL20aa⌬N2 was digested by BsrGI-XbaI, and the DNA fragment carrying L20C was transferred into pET42a (Novagen). Gel Filtration and SAXS Experiments—The complex was formed as described above, by stepwise addition of the RNA to the protein solution, and monitored by NMR. The scattering pattern of a 5 mg/ml solution of bovine serum albumin was recorded, and the value of the intensity at the origin I(0)/c (where c is protein concentration in mg/ml) was used as a reference to derive an estimate of the molecular mass of the samples studied. Contact between the two complexes was detected by monitoring the van der Waals term of XPLOR energy function After these steps, a random dimer conformation was obtained in which the two contacting complexes were related by a 2-fold symmetry axis. This neighboring relationship could be used to partition the set of compliant dimers into disjoint geometric clusters

RESULTS

Target RNA Sequences

Analysis of the Complex Formed with rRNA

The former value is significantly larger than a theoretical

DISCUSSION