Sm-like proteins in Eubacteria: the crystal structure of the Hfq protein from Escherichia coli

Abstract

The Hfq protein was discovered in Escherichia coli in the early seventies as a host factor for the Qbeta phage RNA replication. During the last decade, it was shown to be involved in many RNA processing events and remote sequence homology indicated a link to spliceosomal Sm proteins. We report the crystal structure of the E.coli Hfq protein showing that its monomer displays a characteristic Sm-fold and forms a homo-hexamer, in agreement with former biochemical data. Overall, the structure of the E.coli Hfq ring is similar to the one recently described for Staphylococcus aureus. This confirms that bacteria contain a hexameric Sm-like protein which is likely to be an ancient and less specialized form characterized by a relaxed RNA binding specificity. In addition, we identified an Hfq ortholog in the archaeon Methanococcus jannaschii which lacks a classical Sm/Lsm gene. Finally, a detailed structural comparison shows that the Sm-fold is remarkably well conserved in bacteria, Archaea and Eukarya, and represents a universal and modular building unit for oligomeric RNA binding proteins.

Escherichia coli Hfq has distinct interaction surfaces for DsrA, rpoS and poly(A) RNAs

“…Previous experiments with wild-type Hfq showed what seemed to be mutual binding of A 27 and DsrA to Hfq 37 . In light of the results described above, we carried out a series of competition experiments using gel shift assays to look at the effect of A 27 addition to binary complexes containing either DsrA or rpoS mRNA 5′ UTR prebound to Hfq (Fig.…”

“…Mutations at Tyr25 and Ile30 affected A 27 binding, leading to a ten-fold loss in affinity for Hfq Y25D and Hfq I30D. Double mutants showed no additional changes in affinity for A 27 when Sm2 motif changes were combined with distal face mutations. We therefore infer that poly(A) interacts with the distal face of Hfq.…”

“…Hfq shares sequence similarity to the eukaryotic Lsm proteins [23][24][25][26][27] We addressed these questions through a mutational analysis of Hfq, probing in vitro binding to several model RNAs that represent species with which Hfq must interact. Hfq mutants were assayed in vivo using a reporter assay and RNA lifetime experiments.…”

“…1). Crystallographic characterization of Hfq revealed a classical Sm fold, as predicted from sequence alignment and homology modeling 27,29 . Whereas eukaryotic Sm proteins form heteroheptameric rings30 -32, Hfq forms homo hexamers similar to the archaeal Sm proteins33 ,34 .…”

“…Hfq shares sequence similarity to the eukaryotic Lsm proteins [23][24][25][26][27] . In the Conserved Domain Database 28 Hfq is listed under the Sm and Sm-like protein family as well as among the eubacterial Hfqs.…”