RNase P cleaves transient structures in some riboswitches

Abstract

RNase P from Escherichia coli cleaves the coenzyme B12 riboswitch from E. coli and a similar one from Bacillus subtilis. The cleavage sites do not occur in any recognizable structure, as judged from theoretical schemes that have been drawn for these 5' UTRs. However, it is possible to draw a scheme that is a good representation of the E. coli cleavage site for RNase P and for the cleavage site in B. subtilis. These data indicate that transient structures are important in RNase P cleavage and in riboswitch function. Coenzyme B12 has a small inhibitory effect on E. coli RNase P cleavage of the E. coli riboswitch. Both E. coli RNase P and a partially purified RNase P from Aspergillus nidulans mycelia succeeded in cleaving a putative arginine riboswitch from A. nidulans. The cleavage site may be a representative of another model substrate for eukaryotic RNase P. This 5' UTR controls splicing of the arginase mRNA in A. nidulans. Four other riboswitches in E. coli were not cleaved by RNase P under the conditions tested.

Fig. 1.

Activity of RNase P from E. coli and A. nidulans on various substrates. (A) E. coli btuB 202 mRNA. Reactions were carried out as described in Materials and Methods by using E. coli RNase P holoenzyme with 12,500 cpm of labeled E. coli btuB 202 mRNA substrate. Lane 1, mRNA alone; lanes 2 and 3, mRNA with E. coli RNase P holoenzyme (20 nM M1 RNA/200 nM C5) incubated for 15 and 30 min, respectively, at 37°C; lanes 4 and 5, mRNA with E. coli RNase P holoenzyme (100 nM M1 RNA/1 μM C5) incubated for 15 and 30 min, respectively, at 37°C. (B) A. nidulans 5′ UTR of the arginase gene with controls. Reactions were carried out as described in Materials and Methods by using E. coli RNase P holoenzyme (20 nM M1 RNA/200 nM C5 protein) or A. nidulans concentrated glycerol gradient extracts (2 μl/10 μl reaction) with 10,000 cpm of labeled mRNA substrate as indicated. Reactions were incubated for 20 min at 37°C. Substrates were pTyr, the precursor to Saccharomyces cerevisiae tRNA^Ser (pSupS1), and the A. nidulans mRNA: lane 1, mRNA alone; lane 2, mRNA with E. coli RNase P holoenzyme; and lane 3, mRNA with A. nidulans RNase P extract.

Fig. 2.

Schemes of the E. coli (A) and B. subtilis (B) coenzyme B₁₂ 5′ UTR riboswitches as determined by phylogenetic considerations (1). The RNase P cleavage sites are marked. In A, some inherent uncertainty in the analysis of the RNase T1 digests indicates that the cleavage site might be at G176 rather than at G175. In B. subtilis yvrC, G132 has been changed to C132 to correct the sequence published in ref. . The schemes are reprinted by permission from ref. .

Fig. 3.

Redrawn schemes of btuB regions in E. coli and B. subtilis. Different sequences were chosen, as described in the text, to redraw structures to determine RNase P cleavage sites (7, 8). (A) E. coli btuB 202. (B) The same as in A, except more nucleotides are at the 3′ end, and these give a theoretical structure that has not been tested. (C) B. subtilis yvrc167(btuB). (D) Redrawn from a Zuker structure that showed no RNase P cleavage sites. This has more nucleotides at the 3′ end than does C and represents a possible RNase P cleavage site. The nucleotide numbers refer to those in Fig. 2.

Fig. 4.

Effect of coenzyme B₁₂ on cleavage of E. coli btuB 202 and B. subtilis yvrc167 mRNAs by E. coli RNase P holoenzyme. Reactions were carried out in the dark as described in Materials and Methods by using E. coli RNase P holoenzyme (100 nM M1 RNA/1 μM C5 protein) with 25,000 cpm of labeled E. coli btuB202 or B. subtilis yvrc167 mRNA substrates. For each substrate, lane 1, mRNA alone; lanes 2–4, mRNA with E. coli RNase P holoenzyme, incubated for 10, 20, and 30 min, respectively; and lanes 5–7, same as lanes 2–4 but with the addition of 0.1 mM coenzyme B₁₂ (Sigma).

Fig. 5.

Schematic diagram of pRS414btuB (–70 in the DNA to +450 in frame with the ninth codon of lacZ, a gift of R. Breaker, Yale University) showing locations of the two primers used for Northern analyses. Primer btuB CL covers nucleotides 163–189 of btuB, which includes the site of RNase P cleavage (5′-GCGATGATGAGAACCAGATGCG ACGTTGGC-3′). Primer btuB LZ covers nucleotides 445–450 of btuB (the AUG is located at 241 nucleotides), a linker region between the two genes, and 14 nucleotides of the lacZ gene (5′-ACGTTGTAAAACGACGGG ATCCCGCGGAAG-3′).

Fig. 6.

Scheme of the structure of the arginase mRNA from A. nidulans that shows the RNase P cleavage site. The procedures used were adapted from ref. and show part of the 5′ UTR and of the ORF for arginase. The AUG at the beginning of the ORF is marked, as well as the RNase P cleavage sites [E. coli and A. nidulans (A. nid)].