Identification of a large noncoding RNA in extremophilic eubacteria

Puerta-Fernandez et 10.1073/pnas.0607493103.XXYYYYY103.

Supporting Figures

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Supporting Figure 5
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9

Supporting Figure 5a

Supporting Figure 5b

Fig. 5. Sequence alignment and predicted base-pairing interactions of 35 OLE RNA representatives. Nucleotides shaded in the same color identify stretches of complementary base pairs designated P1-P14. The "Consensus" line, determined by using 35 OLE RNA representatives, identifies nucleotides that exhibit 97% or greater conservation (red) or that exhibit 75% or greater conservation (black). Gray boxes identify the primer binding sites used to PCR amplify OLE RNA sequences from populations of genomic DNAs. Note the following: The main groups of bacteria represented in the environmental samples examined are Chloroflexi, Bacteriodetes, and Proteobacteria. However, organisms belonging to more than 10 different bacterial groups have been identified [Ley RE, Harris JK, Wilcox J, Spear JR, Miller SR, Bebout BM, Maresca JA, Bryant DA, Sogin ML, Pace NR (2006) Appl Environ Microbiol 72:3685-3695]. Although Firmicutes and Actinobacteria were poorly represented in this population, we expected that there would be an enrichment of organisms that have adapted to extreme conditions and that might carry OLE RNAs.

Supporting Figure 6

Fig. 6. Representative in-line probing data for an OLE RNA construct based on the sequence from B. halodurans (ATTC 21591D). To facilitate resolution of cleavage products over the entire length of the RNA, the molecule was divided into sections. Sections of OLE RNA corresponding to nucleotides 449-608 (I), nucleotides 1-291 (II), and nucleotides 284-677 (III) were generated and examined by in-line probing. In lanes designated II and III, the sections of OLE RNA defined were 5' ³²P-labeled, and the remainder of the OLE RNA motif was either withheld (-) or added (+) as unlabeled RNAs in a minimum of 3-fold stoichiometric excess over the labeled section. NR, ^-OH, and T1 contain RNAs that were not reacted, partially digested with alkali, or partially digested with RNase T1 (G-specific cleavage), respectively. Selected bands corresponding to G-specific cleavage are identified by using the numbering system from SI Fig. 7. Regions where the combination of OLE RNA sections causes differences in spontaneous cleavage are boxed. The small number of changes caused by mixing of the two sections suggests that they independently fold to form the secondary structure elements as predicted. The methods were as follows. RNAs were prepared by in vitro transcription of double-stranded DNA products derived by PCR amplification of specific segments of the OLE RNA gene from B. halodurans genomic DNA. RNAs were prepared, purified, 5' ³²P-radiolabeled, and stored by using methods similar to those described previously [Seetharaman S, Zivartz M, Sudarsan N, Breaker RR (2001) Nature Biotechnol 19:336-341]. For each in-line probing reaction, 1 nM 5' ³²P-RNA was incubated for 40-43 h at 25°C in a buffer containing 20 mM MgCl₂, 50 mM Tris•HCl (pH 8.3 at 25°C), and 100 mM KCl. Some reactions as noted also contained an excess of unlabeled molecule to promote intermolecular association. In this case, RNAs were incubated at 65°C for 10 min and then cooled on ice before adding the in-line probing buffer. Products of spontaneous RNA cleavage were resolved by denaturing (8 M urea) 10% PAGE and imaged by using a PhosphorImager.

Supporting Figure 7

Fig. 7. Sequence, secondary structure model, and structural probing of OLE RNA from B. halodurans. Encircled nucleotides undergo substantial levels of spontaneous cleavage during in-line probing assays at internucleotide linkages located 3' of each annotated nucleotide. These annotations were derived from composite data generated by examining the products of in-line probing like that presented in SI Fig. 6. Some locations of spontaneous cleavage hotspots are approximates due to the large size of the fragments under examination.

Supporting Figure 8

Fig. 8. Alignment of amino acid sequences for BH2780 protein and homologous proteins associated with OLE RNAs. Sequences were aligned by using the ClustalW program [Thompson JD, Higgins DG, Gibson TJ (1994) Nucleic Acids Res 22:4673-4680], and a consensus sequence was extracted from this alignment. Symbols in the consensus line are defined as follows: (*), 100% conservation; (:), substitutions are conservative (similar amino acid properties); (.), substitutions are semiconservative (see ClustalW program for details). Shaded boxes identify predicted transmembrane domains.

Supporting Figure 9

Fig. 9. Output of TMHMM prediction for transmembrane domains in the BH2780 protein from B. halodurans. The TMHMM computer algorithm can be accessed on-line at www.cbs.dtu.dk/services/TMHMM.