## 3 Discussion and Conclusions
The asRNA targeting _muoF_ (and the contiguous _muoE_ in some streptomycetes) was hypothesized to play a role as a \"checkpoint\" during complex I assembly [20], while the asRNAs targeting _muoL2_ and _muoM2_ were proposed to regulate the differential assembly of NuoL vs. NuoL2 and NuoM vs. NuoM2 into the respiratory complex I [20], similarly to what was observed in several cyanobacteria in which the multiplicity of functions assigned to cyanobacterial complex I (respiration, cyclic electron flow, and CO2 uptake) relies upon the diversity of the NdhD (NuoM) and NdhF (NuoL) protein isoforms resulting in the occurrence of distinct complex I assemblages [68]. Noteworthy, we found a remarkable number of asRNAs having as putative targets mRNAs of genes involved in translation, ribosomal structure, and biogenesis, in addition, two asRNAs that are transcribed complementary, respectively, to _nusA_, coding for the transcription elongation factor NusA, and _cdnL_, coding for an ANTAR-containing protein. To our knowledge, asRNAs targeting ribosomal protein (r-protein)-encoding genes have not been described so far in _Streptomyces_. However, a survey of small ncRNAs associated with r-protein operons in the bacterial pathogens _Staphylococcus aureus_, _Vibrio cholerae_, _Salmonella enterica_ sv. Typhi, and _Mycobacterium tuberculosis_ reported the presence of a total of 13 asRNAs transcribed complementary to nine r-protein mRNAs [69]. The physiological significance and the molecular mechanism by which these asRNAs can modulate the expression of the r-protein-encoding genes are currently under investigation. In all living organisms, ribosome assembly is subject to extensive feedback regulation to ensure correct ribosome manufacture in response to a variety of environmental and metabolic changes. In bacteria, despite numerous investigations, the mechanisms for the ribosome feedback regulation, and growth rate-dependent regulation of rRNA synthesis in bacteria other than the model organisms _Escherichia coli_ remain to be elucidated. Studies in _E. coli_ demonstrated that r-protein synthesis is tightly regulated by numerous mechanisms including translational coupling, translation repression, or premature transcription termination [70]. In particular, in _E. coli_ expression of more than half of the r-protein genes are controlled by 12 distinct RNA autogenous regulatory elements by which these proteins inhibit the translation of their own mRNA [71]. However, only two or three of these regulatory elements are widely distributed across many bacteria phyla raising some concerns about the full transferability of the r-protein regulation model established in _E. coli_ to other bacteria, including the streptomycetes [71; 72; 73]. The discovery of asRNAs targeting so many r-protein-encoding genes in _S. ambofaciens_ may suggest the existence of an additional regulatory level. In addition to r-protein genes, we detected several asRNAs that are transcribed complementary to the 5 tRNAs were also detected. To our knowledge, this finding is unprecedented in _Streptomyces_. Moreover, it may be interesting to note that one of the 5 tRNAs recognize a specific codon for proline (CCA) that is less represented in _Streptomyces_ CDSs with respect to other codons specifying the same amino acids (Supplementary Figure S2). _Streptomyces_ use rare codons to regulate their physiology. The most known example is _bldA_ that codes for tRNA\({}^{\text{Leu}}\) (UAA) that recognizes a rare leucine codon, UUA (about 0.01% of codons). The tRNA\({}^{\text{Leu}}\) (UAA) is unnecessary for vegetative growth but is required for some aspects of secondary metabolism and morphological development [74]. In streptomycetes, about 3% of genes contain a UUA codon, which is mostly associated with either recently acquired genes by horizontal gene transfer, or genes coding for pleiotropic or pathway-specific regulators of secondary metabolism and morphological development [74].