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Science 329 (5993): 845-848

Copyright © 2010 by the American Association for the Advancement of Science

An Allosteric Self-Splicing Ribozyme Triggered by a Bacterial Second Messenger

Elaine R. Lee1,*, Jenny L. Baker2,*, Zasha Weinberg1,3, Narasimhan Sudarsan1,3, and Ronald R. Breaker1,3,4,{dagger}

1 Department of Molecular, Cellular, and Developmental Biology, Yale University, Box 208103, New Haven, CT 06520–8103, USA.
2 Department of Chemistry, Yale University, Box 208103, New Haven, CT 06520–8103, USA.
3 Howard Hughes Medical Institute, Yale University, Box 208103, New Haven, CT 06520–8103, USA.
4 Department of Molecular Biophysics and Biochemistry, Yale University, Box 208103, New Haven, CT 06520–8103, USA.


Figure 1
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Fig. 1. c-di-GMP-II riboswitches. (A) Consensus sequence and secondary structure model for c-di-GMP-II riboswitch aptamers. Nucleotides in red are conserved in >97% of the representatives. Other annotations are described in fig. S2. (B) Wild-type (WT) 84 Cd RNA encompassing the motif upstream of a possible virulence gene. Disruptive (M2) and restorative (M3) mutations are depicted, and sites of spontaneous cleavage are derived from C. Asterisks identify nucleotides added to facilitate in vitro transcription. (C) Denaturing polyacrylamide gel electrophoresis (PAGE) of WT 84 Cd RNA in-line probing cleavage products. No reaction (NR); partial digestion with ribonuclease (RNase) T1 (T1, cleaves after G residues); alkali (-OH, cleaves at all linkages); incubation in the absence (–) or presence (+) of 10 µM c-di-GMP. Selected RNase T1 digestion product bands are identified. (D) Plot of the fraction of RNA remaining unbound versus the logarithm of the concentration of c-di-GMP present during in-line probing (fig. S3). (E) KD values for 84 Cd binding to c-di-GMP and various di- and mononucleotide analogs. A, adenosine; G, guanosine.

 

Figure 2
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Fig. 2. Architecture, mechanism, and activity of a tandem riboswitch-ribozyme. (A) Tandem c-di-GMP-II aptamer and group I ribozyme arrangement. (B) Key features of the aptamer-ribozyme system, including validated splice and GTP attack sites (fig. S8). Alternative base-pair interactions guiding allosteric function are shaded blue and green. (C) PAGE separation of products generated by self-splicing assays. NR, no reaction; C-IVS and L-IVS, circular and linear intervening sequences, respectively; Pre, 864-nucleotide precursor RNA (fig. S5); 5'E-3'E, spliced exons; 3'E*, 3' fragment generated by GTP attack at an alternative site; Fragments, additional RNA products presumably created by IVS circularization. Data for some mutants is presented elsewhere (fig. S11).

 

Figure 3
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Fig. 3. Rate constant modulation by c-di-GMP. (A) Time course of [{alpha}-32P]GTP attack at sites GTP1 or GTP2 in the absence or presence of c-di-GMP. (B) Plot of the natural logarithm of the fraction of unprocessed or differently processed RNA (pre-dp RNA) versus time for the reaction in (A). Values for fraction processed were corrected for ~50% of the precursor remaining after exhaustive incubation. (C) Time course of the production of spliced exons (5'E-3'E) or alternative GTP2 site fragment (3'E*) in the absence or presence of c-di-GMP. Annotations are as described for Fig. 2C. (D) Plot of the natural logarithm of the fraction of unprocessed or differently processed RNA versus time for the reaction in (C), corrected as described in (B). (E) Changes in splice product yields on introduction of c-di-GMP. (Left) Ratio of the number of 3'E* molecules versus the number of 5'E-3'E spliced exon molecules in the absence or presence of c-di-GMP, respectively. (Right) Ratio of the numbers of 3'E* molecules and the numbers of 5'E-3'E spliced exon molecules in the absence or presence of c-di-GMP, respectively.

 

Figure 4
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Fig. 4. Proposed mechanism for allosteric ribozyme-mediated gene control. (A) Precursor mRNA with the start codon sequestered by the ribozyme P10 stem. (B) RNA processed in the presence of GTP and c-di-GMP unmasks the start codon and creates a perfect ribosome binding site. (C) RNA processed in the presence of GTP alone lacks a ribosome binding site.

 


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