fhlA repression by OxyS RNA: kissing complex formation at two sites results in a stable antisense-target RNA complex.
OxyS is a small untranslated RNA that is induced in response to oxidative stress in Escherichia coli. This small RNA acts as a global regulator affecting the expression of multiple genes. OxyS represses the translation of fhlA, a transcriptional activator for formate metabolism. Previously, we have shown that fhlA repression by OxyS is mediated through base-pairing with a short sequence overlapping the ribosome binding site. Here we show that the OxyS-fhlA interaction involves a second site residing further downstream, within the coding region of fhlA. Mutations that disrupt pairing at this site affect the ability of OxyS to prevent 30 S ribosomes from binding to fhlA mRNA. Structure probing of fhlA mRNA demonstrates that both sites reside in the loops of two stem-loop structures. OxyS-fhlA pairing analysis shows that OxyS binds wild-type fhlA with an apparent dissociation constant of 25 nM, indicating that kissing complex formation between OxyS and fhlA results in a stable antisense-target complex. Mutations at either site, which disrupt pairing of OxyS to fhlA, decrease the stability of this complex. Our results indicate that kissing complex formation is sufficient to repress fhlA translation by OxyS.
Argaman
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L
Department of Molecular Genetics and Biotechnology, The Hebrew University- Hadassah Medical School, Jerusalem, 91120, Israel.
Altuvia
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S
D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D018384:Oxidative Stress / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics; D020871:RNA Stability; D016372:RNA, Antisense / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013463:Sulfuric Acid Esters / Q000378:metabolism; D013816:Thermodynamics; D015534:Trans-Activators / Q000235:genetics*
D029968:Escherichia coli Proteins; D016372:RNA, Antisense; D012329:RNA, Bacterial; D012333:RNA, Messenger; D013463:Sulfuric Acid Esters; D015534:Trans-Activators; C067189:fhlA protein, E coli; C007482:dimethyl sulfate