Abstract
The carbon storage regulator/regulator of secondary metabolism (Csr/Rsm) type of small non-coding RNAs (sRNAs) is widespread throughout bacteria and acts by sequestering the global translation repressor protein CsrA/RsmE from the ribosome binding site of a subset of mRNAs. Although we have previously described the molecular basis of a high affinity RNA target bound to RsmE, it remains unknown how other lower affinity targets are recognized by the same protein. Here, we have determined the nuclear magnetic resonance solution structures of five separate GGA binding motifs of the sRNA RsmZ of Pseudomonas fluorescens in complex with RsmE. The structures explain how the variation of sequence and structural context of the GGA binding motifs modulate the binding affinity for RsmE by five orders of magnitude (∼10 nM to ∼3 mM, Kd). Furthermore, we see that conformational adaptation of protein side-chains and RNA enable recognition of different RNA sequences by the same protein contributing to binding affinity without conferring specificity. Overall, our findings illustrate how the variability in the Csr/Rsm protein–RNA recognition allows a fine-tuning of the competition between mRNAs and sRNAs for the CsrA/RsmE protein.
Highlights
Regulation by small non-coding RNAs is crucial for orchestrating global changes in bacterial gene expression [1,2,3]
The first RsmE protein dimer binds simultaneously to GGA#3 and GGA#5, whereas a second RsmE dimer binds to GGA#4, which is the weakest binding motif. These findings suggest that one RsmE dimer binding to both GGA#3 and GGA#5, which overlaps with the ribosome binding site (RBS), is responsible for translation repression of the hcnA messenger RNAs (mRNAs)
We have elucidated and compared the solution complex structures of RsmE bound to six different target RNAs, which are the four SLs and the single-stranded region between SL2 and SL3 of the small non-coding RNAs (sRNAs) RsmZ and a 20 nt RNA SL encompassing the RBS of the hcnA mRNA in P. fluorescens [11]
Summary
Regulation by small non-coding RNAs (sRNAs) is crucial for orchestrating global changes in bacterial gene expression [1,2,3]. The best studied small regulatory RNAs in bacteria function by Hfq chaperone-assisted base pairing with target messenger RNAs (mRNAs) [4,5] They often work by binding to the ribosome binding site (RBS), thereby repressing mRNA translation and through recruiting RNase E via Hfq, they can target the mRNA for degradation [6]. Another important group of sRNAs do not act on mRNAs directly but function by sequestering the CsrA-type protein from the RBS of a subset of mRNAs and activate translation initiation [1,7,8]. The de-repressor sRNAs contain several GGA binding motifs, typically located in hairpin loops [14,18,19,20,21], in single-stranded regions or even buried within secondary structures
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