7242 Nucleic Acids Research, 2008, Vol. 36, No. 22 RNA isolation and Northern blot analysis RNA was isolated as described in Hopwood et al. (31) with few modiﬁcations. Cultures were grown on the sur- face of cellophane discs overlaying solid agar medium. At various timepoints, cultures were harvested by scraping cells into modiﬁed Kirby’s mixture [1% w/v N-lauroylsar- cosine sodium salt, 6% w/v sodium 4-amino salycilate, 6% v/v phenol mixture (pH 7.9) made in 50 mM Tris (pH 8.3)]. UV spectroscopy and agarose gel electrophor- esis were used to assess the quantity and quality of total RNA samples. Northern blots were prepared as described previously (30), only after hybridizing with appropriate 50 end-labelled oligonucleotide probes (see Supplementary Table S2 for sequences), membranes were washed twice for 5 min with 2 SSC, 0.1% SDS, followed by a ﬁve minute high stringency wash with 0.2 SSC, 0.1% SDS. Northern blots for all detectable sRNAs were conducted a minimum of three times, using RNA isolated from at least two independent time courses. Comparisons between sRNA transcripts in wild-type and bld/whi mutants were done using RNA transferred to the same (single) mem- brane, hybridized to the same labelled oligonucleotide probe, and exposed to X-ray ﬁlm for the same length of time. This ensured that wild-type and mutant RNA sam- ples were treated equivalently at all stages, and thus any diﬀerences observed could be attributed to diﬀering levels of sRNA transcript, and not to inconsistent probe label- ling or diﬀering exposure times. Mapping of 5’- and 3’-ends 50-rapid ampliﬁcation of cDNA ends (RACE) was carried out using the FirstChoice RLM-RACE kit (Ambion), following the manufacturer’s instructions, but adjusted for use with bacterial RNA. Brieﬂy, 10 mg of total RNA, extracted from S. coelicolor M600 at speciﬁc timepoints, was treated with tobacco acid pyrophosphatase (TAP) before being ligated to a 50-RACE adapter (see Supple- mentary Table S2) using T4 RNA ligase. This ligated product was then used as template for reverse transcrip- tion using a primer complementary to the sRNA sequence (Supplementary Table S2), together with SuperScriptTM III reverse transcriptase (Invitrogen). The resulting cDNA then served as template for 50-end PCR ampliﬁca- tion, using an adaptor-speciﬁc oligonucleotide and the same oligonucleotide used to prime the reverse transcrip- tion reaction (Supplementary Table S2). The resulting PCR product was then used as template for a second reac- tion, involving the use of a second ‘nested’ adaptor- speciﬁc oligonucleotide, and a nested sRNA-speciﬁc oligonucleotide (Supplementary Table S2). The PCR pro- ducts from the second reaction were separated on an agarose gel, excised, and puriﬁed using the Qiagen MinElute PCR puriﬁcation kit. The puriﬁed products were cloned into the TOPO vector using TOPO TA Cloning (Invitrogen) and transformed into One Shot DH5a-TOP10F’ competent cells (Invitrogen). Colony PCR, using M13 Fwd and Rev primers (Supplementary Table S2), was conducted to identify those colonies containing cDNA inserts, which were then grown over- night. Plasmid DNA was extracted using the QIAprep Miniprep kit, and the cloned inserts were sequenced (Sup- plementary Table S3). 30-RACE was conducted using the same kit as the 50-RACE, following the manufacturer’s instructions. Prior to initiating the 30-RACE protocol, however, 40 mg of total RNA was polyadenylated by treatment with 5 U of poly(A) polymerase (2 U/ml, Ambion) at 378C for 1 h. The 30-RACE adapter was then annealed to 50 ng poly(A)-RNA, and this RNA was reverse transcribed using M-MLV reverse transcriptase (Ambion) for 1 h at 428C. PCR, cloning and sequencing were conducted as described for 50-RACE (Supplementary Table S3). In instances where it was not possible to map the 50- or 30-ends, northern blot analysis was used to determine the approximate transcript ends. Overlapping oligonucleotide probes were individually hybridized to northern blots, and start and end sites were mapped to within 10 nt. RESULTS Comparative genomic and computational predictions of candidate sRNA genes in S. coelicolor Bioinformatic prediction of trans-encoded sRNAs in S. coelicolor is hindered by a lack of readily detectable promoter and transcription terminator sequences, but is greatly facilitated by the availability of multiple actinomy- cete genome sequences for use in comparative genomic analyses. We therefore opted to conduct a comparative genomics-based search for sRNAs in S. coelicolor, using sequence conservation in other actinomycetes as our start- ing point. We took advantage of the fact that actinomy- cetes appear to share a conserved genetic ‘core’ (2), and focussed our search on large intergenic (IG) sequences (>200 nt) within this core region. In S. coelicolor, the con- served core covers 4.9 Mb of its linear genome and is centrally positioned, housing most essential (house- keeping) genes. The left and right ‘arm’ regions on either side of the conserved core extend 1.5 and 2.3 Mb, respec- tively, and contain primarily non-essential, S. coelicolor- speciﬁc genes, including those for most secondary metabolic pathways (2). There were 833 IG regions larger than 200 nt within the conserved core region, and 169 of these regions (20.29%) were unique to S. coelicolor. The remaining 664 IG regions contained sequences that were conserved (E-value <0.001) in other organisms (Supplementary Table S1), with the majority of these (520 or 62.42% overall) having similarity to sequences in other streptomycetes. A small proportion of IG regions (6.12%) contained sequences with similarity to those in other actinomycetes, and included sequences corresponding to degenerate transposases, tRNAs and predicted riboswitches (e.g. thiamine pyrophosphate riboswitch). Beyond the actinomycetes, sequences within 68 IG regions (8.16%) were conserved in more divergent bacterial species, and again, included predominantly tRNA genes (>50% of conserved sequences), and other well characterized sequences such as the RNase P gene (32) and the FMN-sensing riboswitch (33). Finally, a small number of sequences had similarity to eukaryotic sequences (3%), including those from Homo sapiens and