<i>psm-mec</i> RNA specifically binds <i>agrA</i> mRNA and inhibits its translation.

Abstract

<p>(<b>A</b>) Hybridization between <i>psm-mec</i> RNA and <i>agrA</i> mRNA was predicted by an <i>in silico</i> program RNA hybrid. Black and gray lines represent strong and weak hydrogen bonds, respectively. (<b>B</b>) Binding between <i>psm-mec</i> RNA and <i>agrA</i> RNA (−20–717) was analyzed using a gel-retardation assay. Various amounts of nonlabeled <i>agrA</i> RNA were added to ³²P-labeled <i>psm-mec</i> RNA (0.13 pmol), and electrophoresed in 6% native polyacrylamide gel. In the right six lanes, nonlabeled <i>psm-mec</i> RNA or yeast tRNA was added to compete with the binding between <i>agrA</i> RNA and ³²P-labeled <i>psm-mec</i> RNA. (<b>C</b>) Binding experiment between <i>psm-mec</i> RNA and deletion mutants of <i>agrA</i> RNA. Various amounts of nonlabeled <i>agrA1</i> RNA (−20–267) or <i>agrA2</i> RNA (−20–198) were added to ³²P-labeled <i>psm-mec</i> RNA (0.13 pmol). (<b>D</b>) Nucleotide sequences of <i>psm-mec</i> RNA, a deletion mutant of <i>psm-mec</i> RNA (<i>psm-mec-</i>D), and a nucleotide-substituted <i>psm-mec</i> RNA (<i>psm-mec-</i>M) are presented. Red dotted line in <i>psm-mec</i>-D indicates the deleted region. Red letters in <i>psm-mec-</i>M indicate the substituted nucleotides that are not complementary to <i>agrA</i> RNA. (<b>E</b>) Various amounts of nonlabeled <i>psm-mec</i> RNA, <i>psm-mec-</i>D RNA, or <i>psm-mec-</i>M RNA were added to ³²P-labeled <i>agrA1</i> RNA (−20–267), and electrophoresed in 6% native polyacrylamide gel. (<b>F</b>) Luciferase activities of Newman strains that were transformed with pGP-agrA-luc carrying no <i>psm-mec</i> (−F), <i>psm-mec</i> (+F), <i>psm-mec</i>-D, or <i>psm-mec</i>-M were measured. The vertical axis represents the relative luciferase activity against that of pGP-agrA-luc carrying no <i>psm-mec</i>. Means ± standard deviations from three independent experiments are presented. Student t-test P-values are presented. (<b>G</b>) Cell extracts (3 µg protein) of 24 h-cultures of Newman strains transformed with pND50 (empty vector), pF carrying <i>psm-mec</i>, p-psm-mec-D carrying <i>psm-mec-</i>D, or p-psm-mec-M carrying <i>psm-mec-</i>M were subjected to Western blotting by anti-AgrA IgG (Left panel). Band intensities of AgrA were measured (Right graph). Means ± standard deviations from three independent experiments are presented. Student t-test P-values are presented. (<b>H</b>) Amounts of PSMα3 in the supernatants of 24 h-cultures of Newman strains transformed with <i>psm-mec</i>, <i>psm-mec-</i>D, or <i>psm-mec</i>-M were measured. The vertical axis represents the relative amount of PSMα3 against that of Newman strain transformed with pND50 (empty vector). Student t-test P-values are presented.</p