1887

Abstract

The 60 nucleotides encoding the signal peptide of the membrane-bound lytic transglycosylase (MltA) homologue GNA33 were found to exert a negative regulatory effect on expression of from either a T7- or a P-driven system in . Down-regulation was observed to occur at the transcriptional/post-transcriptional level and could possibly be ascribed to the formation of a stem–loop secondary structure within the signal peptide sequence. Slowing down the transcription rate through inhibition/titration of the RNA polymerase resulted in a considerable increase in mRNA accumulation, suggesting that a better coupling of translation to transcription would impede the formation of the putative secondary structure. Screening of synonymous mutations in the signal peptide sequence that showed high-level expression of an in-frame fusion to a reporter resulted in the isolation of several deletion mutants lacking most of the sequence participating in the putative secondary structure. Interestingly, the increase in the steady-state mRNA level observed in deletion mutants was higher, reaching a 300-fold increment, than that found in substitution mutants. Our results support the hypothesis that the rate of transcription controls the formation of a secondary structure in the region of the transcript corresponding to the signal peptide sequence and this, when formed, negatively regulates expression.

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2004-05-01
2020-03-29
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