RNA pentaloop structures as effective targets of regulators belonging to the RsmA/CsrA protein family

Abstract

In the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens CHA0, the dimeric RNA-binding proteins RsmA and RsmE, which belong to the vast bacterial RsmA/CsrA family, effectively repress translation of target mRNAs containing a typical recognition sequence near the translation start site. Three small RNAs (RsmX, RsmY, RsmZ) with clustered recognition sequences can sequester RsmA and RsmE and thereby relieve translational repression. According to a previously established structural model, the RsmE protein makes optimal contacts with an RNA sequence 5′-^A/_UCANGGANG^U/_A-3′, in which the central ribonucleotides form a hexaloop. Here, we questioned the relevance of the hexaloop structure in target RNAs. We found that two predicted pentaloop structures, AGGGA (in pltA mRNA encoding a pyoluteorin biosynthetic enzyme) and AAGGA (in mutated pltA mRNA), allowed effective interaction with the RsmE protein in vivo. By contrast, ACGGA and AUGGA were poor targets. Isothermal titration calorimetry measurements confirmed the strong binding of RsmE to the AGGGA pentaloop structure in an RNA oligomer. Modeling studies highlighted the crucial role of the second ribonucleotide in the loop structure. In conclusion, a refined structural model of RsmE-RNA interaction accommodates certain pentaloop RNAs among the preferred hexaloop RNAs.

Keywords: :

• RNA-binding protein
• CsrA/RsmA
• translational regulation
• Shine-Dalgarno structure
• Gac/Rsm pathway

View correction statement:

Correction to RNA Biology Volume 10, Issue 6

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by the Sandoz Family Foundation (Programme for academic promotion) to K.L. D.H. is grateful to the Swiss National Science Foundation for support. We thank Nicolas Pradervand for performing RsmE cross-linking experiments, Dr Michel Cuendet for his help in developing the RNA parameters for Gromacs, Prof. Shamoo from the Rice University, for kindly providing the Linux version of the Entangle program and the VITAL-IT group of the Swiss Institute of Bioinformatics for providing computational resources. The bioinformatics aspects of this work were performed at the Protein Modeling Facility of the Lausanne University.