The following supplementary material is available for this article: Appendix S1. Material and methods. Table S1. Sequence of the cDNA clones recovered from L7Ae immunocomplexes in S. solfataricus. From a total of 128 insert containing clones, 45 distinct sequences have been identified and organized into six major groups based on the presence of known sequence and/or structural motifs and on their genomic location. The rRNA or tRNA fragments obtained in the library are not shown. Conserved sequence elements corresponding to C, D', C', D in C/D box RNAs and the ACA in H/ACA box RNAs are boxed. Guide regions with identifiable RNA targets are highlighted in grey. The C/D box representatives are aligned using the boxes as anchor; dashed lines correspond to gaps in the alignment. The region of sR117 complementary to the D box guide of sR106, is highlighted. The cDNA sequence overlapping either the sense or the antisense (italics) strands of annotated ORFs is shown in bold. The positions of the initiation and termination codons are highlighted and underlined, respectively. Table S2. Functional homologues of S. acidocaldarius C/D box RNAs in the related genomes of S. solfataricus and S. tokodaii. The nomenclature of the small RNAs is based on the initial set of 28 sequences identified in S. acidocaldarius (Omer et al., 2000 and unpublished results). Sac, S. acidocaldarius, Sto, S. tokodaii, Sso, S. solfataricus, Ssh, S. shibatae; C, D', C', D elements are boxed and the guide region complementary to rRNA or tRNA is underlined in dashed line; sequence alignments were performed using the box elements as anchors where dashed lines indicate gaps in the alignments. For Sso sR9, a B is used to distinguish the respective Sso sRNA from the one having the same identification number previously deposited in the Gene bank (Omer et al., 2000). Fig. S1. Sucrose gradient sedimentation of particles containing the ribosomal protein L7Ae from Sulfolobus solfataricus cell extracts. A S. solfataricus lysate was layered onto a 10%-30% sucrose gradient and sedimented in a SW27 rotor (10?C, 18 K, 16 hours). Fractions (1.5ml) were collected from top to bottom of the gradient. (A) Western blot analysis was performed on an aliquot from every second fraction using S. solfataricus anti-L7Ae antibodies. The position of the cellular L7Ae polypeptide throughout the gradient is indicated. (B) A second aliquot (200ml) from every second fraction were subjected to immunoaffinity chromatography using S. solfataricus anti-L7Ae sera. The RNA present in the immunoprecipitates was recovered by phenol extraction, pCp end-labelled and separated on an 8% denaturing polyacrylamide gel and visualized by autoradiography as described by Omer et al. (2000). The sizes of predominant RNA species are indicated on the right. Fig. S2. Sequence conservation of the atypical C/D box RNAs sR107. DNA sequence alignment of the region spanning the sR107 cDNA, with conserved regions in present in the genomes of S. tokodaii, T. volcanium and with two less related regions in the genome of S. solfataricus. The size of the sequence used in the alignment corresponds most probably to the length of the encoded in vivo transcript (see text). Gaps in the alignment are indicated by dashed line; the 5'-end of the sR107 cDNA is shown by black arrowhead. In S. solfataricus the sR107 cDNA partially overlaps the annotated transposase 1974; a homologue of transposase 1974 is present in the S. tokodaii genome; in T. volcanium, a non-protein coding, intergenic region, designated TvoINTG, exhibits a high degree of sequence conservation; two additional regions in S. solfataricus appear related to the sR107 encoding DNA: one encodes for an inactivated form of transposase 1974 and the other is situated in a non-protein coding region. The corresponding amino acid sequence of the S. solfataricus and S. tokodaii transposase 1974 homologues is shown above the DNA alignment; the translation frame of the inactivated form of transposase 1974 is indicated below the alignment. In the alignment, D'-like and D-like boxes are in bold and underlined. Fig. S3. Intergenic RNAs. (A) sR124 RNA is an intermediate resulted by ligation of the processed pre-rRNA spacers. Proposed secondary folding of sR124 RNA, adapted from Tang et al., (2002b).The possible K-turn formed by juxtaposition of the C'- and D-like boxes is boxed. The position of the endonucleolytic cleavage that liberates the 23S rRNA subunit is indicated by arrow. (B) sR125 RNA contains a K-turn motif (boxed) and is able to bind recombinant L7Ae protein (see text).

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