Structural mechanism of Staphylococcus aureus Hfq binding to an RNA A-tract

Abstract

Hfq is a post-transcriptional regulator that plays a key role in bacterial gene expression by binding AU-rich sequences and A-tracts to facilitate the annealing of sRNAs to target mRNAs and to affect RNA stability. To understand how Hfq from the Gram-positive bacterium Staphylococcus aureus (Sa) binds A-tract RNA, we determined the crystal structure of an Sa Hfq–adenine oligoribonucleotide complex. The structure reveals a bipartite RNA-binding motif on the distal face that is composed of a purine nucleotide-specificity site (R-site) and a non-discriminating linker site (L-site). The (R–L)-binding motif, which is also utilized by Bacillus subtilis Hfq to bind (AG)3A, differs from the (A–R–N) tripartite poly(A) RNA-binding motif of Escherichia coli Hfq whereby the Sa Hfq R-site strongly prefers adenosine, is more aromatic and permits deeper insertion of the adenine ring. R-site adenine-stacking residue Phe30, which is conserved among Gram-positive bacterial Hfqs, and an altered conformation about β3 and β4 eliminate the adenosine-specificity site (A-site) and create the L-site. Binding studies show that Sa Hfq binds (AU)3A ≈ (AG)3A ≥ (AC)3A > (AA)3A and L-site residue Lys33 plays a significant role. The (R–L) motif is likely utilized by Hfqs from most Gram-positive bacteria to bind alternating (A–N)n RNA.