Abstract
Threonylcarbamoyladenosine is a universally conserved essential modification of tRNA that ensures translational fidelity in cellular milieu. TsaD, TsaB and TsaE are identified as tRNA-A37-threonylcarbamoyl (t6A)-transferase enzymes that have been reconstituted in vitro, in few bacteria recently. However, transcriptional organization and regulation of these genes are not known in any of these organisms. This study describes the intricate architecture of a complex multicistronic alr-groEL1 operon, harboring essential genes, namely tsaD, tsaB, tsaE, groES, groEL1, and alr (required for cell wall synthesis), and rimI encoding an N-α- acetyltransferase in Mycobacterium tuberculosis. Using northern blotting, RT-PCR and in vivo fluorescence assays, genes alr to groEL1 were found to constitute an ~6.3 kb heptacistronic operon with multiple internal promoters and an I-shaped intrinsic hairpin-like cis-regulatory element. A strong promoter PtsaD within the coding sequence of rimI gene is identified in M. tuberculosis, in addition. The study further proposes an amendment in the known bicistronic groESL1 operon annotation by providing evidence that groESL1 is co-transcribed as sub-operon of alr-groEL1 operon. The architecture of alr-groEL1 operon, conservation of the genetic context and a mosaic transcriptional profile displayed under various stress conditions convincingly suggest the involvement of this operon in stress adaptation in M. tuberculosis.
Highlights
Survival of a cell depends on its ability to maintain translational fidelity and accuracy
For the reasons that: (a) most genes in this cluster are essential for survival or virulence of M. tuberculosis, (b) they exist in an interesting context with a protein N-α-acetyltransferase, (c) the transcriptional organization and regulation of tRNA-A37-t6A transferase pathway is not known in bacteria yet, and d) the tRNA-A37-t6A transferase machinery is differently organized across different bacterial species, we decided to investigate this genetic context at the transcriptional level
Physical clustering of the encoding genes is observed in the genomes of several bacteria in different contexts, yet no experimental data is available on transcriptional organization and regulation of such an important and conserved pathway except for Neisseria gonorrhoeae wherein tsaD is shown to be co-regulated with cytochrome genes[35]
Summary
Survival of a cell depends on its ability to maintain translational fidelity and accuracy. In M. tuberculosis, the homologs of tRNA-A37-t6A transferase machinery are present in a neighborhood where tsaD (Rv3419c), tsaB (Rv3421c) and tsaE (Rv3422c) are clustered together with rimI (Rv3420c) encoding an N-α-acetyltransferase and alr (Rv3423c) that codes for a well-characterized essential enzyme alanine racemase (Supplementary Figure S1). For the reasons that: (a) most genes in this cluster are essential for survival or virulence of M. tuberculosis, (b) they exist in an interesting context with a protein N-α-acetyltransferase, (c) the transcriptional organization and regulation of tRNA-A37-t6A transferase pathway is not known in bacteria yet, and d) the tRNA-A37-t6A transferase machinery is differently organized across different bacterial species, we decided to investigate this genetic context at the transcriptional level
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