Evidence for the translational attenuation model: ribosome-binding studies and structural analysis with an in vitro run-off transcript of ermC.
Several features of the translational attenuation model of ermC regulation were tested. This model predicts two possible secondary structures for the leader of the ermC transcript and requires that the leader contains two Shine-Dalgarno (SD) sequences. The ribosome binding site for a leader peptide (SD1) is predicted to be accessible, whereas that for the rRNA methylase protein that confers erythromycin (Em) resistance (SD2) is sequestered by base pairing. The model suggests that in the presence of inducer (Em), a ribosome stalls while translating the peptide, altering the mRNA conformation, thereby exposing SD2. The results of our ribosome binding studies demonstrate that SD1 is exposed and binds to ribosomes, whereas SD2 is unavailable. Also, the secondary structure of the 5' region of the ermC transcript was analyzed using methidium propyl-EDTA.Fe (II), T1 nuclease, and nucleases from cobra venom and mung bean sprouts as structure probes. Our results support the previously proposed model for folding of ermC mRNA, and demonstrate that SD1 is single-stranded, while SD2 and its neighboring sequences are largely base paired, consistent with the ribosome-binding results.
28 references, click to expand
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