7248 Nucleic Acids Research, 2008, Vol. 36, No. 22 expressed more highly on one medium type than the other. The most extreme examples of this were seen for scr1906, scr3261 and 3287, which were expressed exclusively under one culture condition. Such media-speciﬁc expres- sion is unusual but not unprecedented in S. coelicolor, with the best studied example being the ram genes, which play an important role in aerial hyphae formation (40) and are expressed exclusively on rich medium (41). Several of the identiﬁed sRNAs appear to be expressed at relatively constitutive levels throughout development (excepting late sporulation), and not surprisingly, these were unaﬀected by developmental mutations. We exam- ined the expression of all sRNAs in two diﬀerent bld mutants: bldB (mutant in which the bldB gene, encoding a protein of unknown function, has been deleted), and bldA (mutant carrying a point mutation in the anticodon of the bldA-encoded leucyl tRNA). The bldB deletion had a variable eﬀect on the expression of four diﬀerent sRNAs, while the bldA mutation consistently resulted in increased expression of three diﬀerent sRNAs. scr1906 expression on rich medium in the bld mutants is particu- larly noteworthy. The observed loss of media dependence for scr1906 expression in both bld mutants may be the consequence of defective catabolite repression—a pheno- type associated with many bld mutations, including bldA and bldB (42). Previous work has also demonstrated that RNaseE activity is upregulated during S. coelicolor devel- opment, and that this upregulation depends upon a func- tional bldA gene product (43). The increased expression seen for scr1906, scr2101 and scr4677 (and the diﬀerent transcript ratios observed for scr3558) in a bldA mutant suggests that these sRNAs, and possibly their mRNA targets, may be degraded by RNaseE, and that they may be stabilized in the presence of a bldA mutation. Interest- ingly, the bldA mutant eﬀects were primarily observed during growth on rich medium; it will be interesting to determine whether RNaseE activity is in any way depen- dent upon growth medium composition. The most signiﬁcant eﬀect of either whi mutation was the complete loss of scr1906 expression in a whiG mutant. whiG encodes the sporulation sigma factor sWhiG, and thus may be responsible for directing the transcription of scr1906. The promoter sequence recognized by sWhiG has been deﬁned, as its control of two other whi genes, whiH and whiI, has been well characterized (44,45). An examination of the scr1906 promoter region did not reveal a sWhiG-like recognition sequence [T(A/C)AA (N)16 GCCGA] (45), suggesting instead that the sWhiG dependence might be an indirect eﬀect. Both whiH and whiI encode DNA-binding transcription factors (44,45), and thus it is possible that one of these two gene products is needed for scr1906 expression. We are currently work- ing to test this model. The existence of scr3558 has been predicted twice pre- viously, once in an earlier sRNA screen of the S. coelicolor genome (39), and once in a comparative genomic search for structured RNAs in bacteria (46). In the latter instance, this region was shown to be conserved within the actinobacteria (including Streptomyces, Corynebacter- ium, Mycobacterium, Frankia and Nocardia), and was termed the ‘6C’ RNA, due to six conserved cytosine residues found in the loop region of a conserved stem- loop structure (46). It was, however, not clear whether this ‘6C RNA’ represented a non-coding extension of an adjacent coding sequence, or whether it was an indepen- dent sRNA. Our work here conﬁrms that distinct sRNA species are indeed encoded within this region. We detected three transcripts, each of which had a diﬀerent 50- and 30-end (as determined by sequential northern blot analysis using overlapping primers). This could mean that each transcript initiates from its own promoter, or that one or both of the two shorter transcripts is processed from a larger transcript. The expression proﬁle of the two shorter transcripts in particular, suggests that they are upregulated later in development, at a time that corre- sponds to spore formation. Not all actinobacteria in which this sequence is conserved, however, form spores, so the role of scr3558/6C RNA may instead reﬂect a gen- eral dormancy or metabolic slow-down response. The location of scr4677 and 3287 suggest potential regulatory targets for both sRNAs. In the case of scr4677, transcription initiates only 15 nt upstream of the SCO4676 start codon (in a divergent orientation), suggest- ing that its product may interact with the 50 untranslated region of SCO4676, possibly modulating transcript stabi- lity. SCO4676 encodes a protein of unknown function that is conserved in other Streptomyces species. For a3287, its primary target would be expected to be SCO3287, the ﬁrst gene of a four gene operon, which encodes a product bearing no sequence similarity to any known protein. Preliminary investigations into SCO3287 expres- sion proﬁles have, however, revealed extremely low trans- cript levels in wild-type strains at all times during development (Hindra and Elliot, M.A., unpublished data). Interestingly, several sRNA sequences appear to be duplicated, or repeated, within the S. coelicolor genome (Figure 5). Based upon comparisons with S. avermitilis and S. griseus, the sequence encompassing scr3045, together with the adjacent genes SCO3045 and SCO3046, appears to have been duplicated in S. coelicolor, generating scr3043, SCO3043 and SCO3044 (Figure 5A), as these sequences are all present only once in the other Strepto- myces species. We detected transcripts from both sRNA regions using 30-end mapping (the two transcripts could not be diﬀerentiated by northern blotting); however, only the 50-end of the scr3045 transcript was successfully mapped. As shown in Figure 5A, the transcript is predicted to be highly structured. There also appear to be two copies of scr3974 in S. coelicolor, with the second located between SCO3869 and SCO3870 (Figure 5B). These two regions were examined using diﬀerential northern blotting, which, along with 50- and 30-transcript end mapping, suggested that only the scr3974 region was expressed. As for scr3045, there is only a single copy of the scr3974 gene found in S. avermitilis and S. griseus, at a site adjacent to the SCO3974 orthologue. Investigation of scr3261 expres- sion also required careful analysis, as it bore a striking resemblance to a sequence within the SCO5333–SCO5334 intergenic region (Figure 5C). The scr3261 sequence contains an enormous stem loop structure, with the stem itself being 66 nt long (Figure 5C); this feature is also conserved in the SCO5333–5334 region. Northern blots