Translational activation of rpoS mRNA by the non-coding RNA DsrA and Hfq does not require ribosome binding.
At low temperature, translational activation of rpoS mRNA, encoding the stationary phase sigma-factor, sigma(S), involves the small regulatory RNA (sRNA) DsrA and the RNA chaperone Hfq. The Hfq-mediated DsrA-rpoS interaction relieves an intramolecular secondary structure that impedes ribosome access to the rpoS ribosome binding site. In addition, DsrA/rpoS duplex formation creates an RNase III cleavage site within the duplex. Previous biochemical studies suggested that DsrA and Hfq associate with the 30S ribosomal subunit protein S1, which implied a role for the ribosome in sRNA-mediated post-transcriptional regulation. Here, we show by ribosome profiling that Hfq partitions with the cytoplasmic fraction rather than with 30S subunits. Besides, by employing immunological techniques, no evidence for a physical interaction between Hfq and S1 was obtained. Similarly, in vitro studies did not reveal a direct interaction between DsrA and S1. By employing a ribosome binding deficient rpoS mRNA, and by using the RNase III clevage in the DsrA/rpoS duplex as a diagnostic marker, we provide in vivo evidence that the Hfq-mediated DsrA/rpoS interaction, and consequently the structural changes in rpoS mRNA precede ribosome binding. These data suggest a simple mechanistic model in which translational activation by DsrA provides a translationally competent rpoS mRNA to which 30S subunits can readily bind.
Vecerek
Branislav
B
Max F. Perutz Laboratories, Department of Microbiology, Immunobiology and Genetics, University of Vienna, Dr Bohrgasse 9, 1030 Vienna, Austria.
Beich-Frandsen
Mads
M
Resch
Armin
A
Bläsi
Udo
U
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D015153:Blotting, Western; D003593:Cytoplasm / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D035001:Host Factor 1 Protein / Q000032:analysis / Q000378:metabolism*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry; D058727:RNA, Small Untranslated; D022661:RNA, Untranslated / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012808:Sigma Factor / Q000096:biosynthesis / Q000235:genetics*
D001426:Bacterial Proteins; C405431:DsrA RNA, E coli; D029968:Escherichia coli Proteins; C087373:Hfq protein, E coli; D035001:Host Factor 1 Protein; D012333:RNA, Messenger; D058727:RNA, Small Untranslated; D022661:RNA, Untranslated; D012269:Ribosomal Proteins; D012808:Sigma Factor; C544829:ribosomal protein S1, E coli; C069567:sigma factor KatF protein, Bacteria