Mechanism for gene control by a natural allosteric group I ribozyme (original) (raw)

1. Narasimhan Sudarsan1,2 and
2. Ronald R. Breaker1,2,3,4
3. 1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA
4. 2Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520, USA
5. 3Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA

Abstract

An allosteric ribozyme consisting of a metabolite-sensing riboswitch and a group I self-splicing ribozyme was recently found in the pathogenic bacterium Clostridium difficile. The riboswitch senses the bacterial second messenger c-di-GMP, thereby controlling 5′-splice site choice by the downstream ribozyme. The proximity of this allosteric ribozyme to the open reading frame (ORF) for CD3246 suggests that coenzyme-mediated regulation of splicing controls expression of this putative virulence gene. In the presence of c-di-GMP, the allosteric ribozyme in the CD3246 precursor transcript generates a spliced transcript that retains the riboswitch aptamer. In the absence of c-di-GMP, the ribozyme mediates an alternative GTP attack that results in a truncated transcript (alternative GTP-attack product). Using reporter assays in Escherichia coli, we investigated the difference in gene expression between the spliced product and the alternative GTP-attack product. We provide evidence that CD3246 gene expression is activated if allosteric ribozyme splicing creates a ribosome binding site (RBS) for translation from a UUG start codon. In addition, biochemical and genetic analyses reveal that the riboswitch may further control CD3246 expression by revealing or occluding this newly formed RBS. Therefore, this architecture provides the riboswitch with a mechanism for extended regulation after splicing has occurred or as a backup mechanism for suppression of translation in the event of misregulated splicing.

• c-di-GMP
• noncoding RNA
• riboswitch
• second messenger
• self-splicing

Footnotes

• ↵4 Corresponding author.
  E-mail ronald.breaker{at}yale.edu.

• Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2757311.

• Received March 29, 2011.

• Accepted August 19, 2011.

• Copyright © 2011 RNA Society

Freely available online through the RNA Open Access option.