Abstract
Efficient and highly organized regulation of transcription is fundamental to an organism’s ability to survive, proliferate, and quickly respond to its environment. Therefore, precise mapping of transcriptional units and understanding their regulation is crucial to determining how pathogenic bacteria cause disease and how they may be inhibited. In this study, we map the transcriptional landscape of the bacterial pathogen Streptococcus pneumoniae TIGR4 by applying a combination of high-throughput RNA-sequencing techniques. We successfully map 1864 high confidence transcription termination sites (TTSs), 790 high confidence transcription start sites (TSSs) (742 primary, and 48 secondary), and 1360 low confidence TSSs (74 secondary and 1286 primary) to yield a total of 2150 TSSs. Furthermore, our study reveals a complex transcriptome wherein environment-respondent alternate transcriptional units are observed within operons stemming from internal TSSs and TTSs. Additionally, we identify many putative cis-regulatory RNA elements and riboswitches within 5’-untranslated regions (5’-UTR). By integrating TSSs and TTSs with independently collected RNA-Seq datasets from a variety of conditions, we establish the response of these regulators to changes in growth conditions and validate several of them. Furthermore, to demonstrate the importance of ribo-regulation by 5’-UTR elements for in vivo virulence, we show that the pyrR regulatory element is essential for survival, successful colonization and infection in mice suggesting that such RNA elements are potential drug targets. Importantly, we show that our approach of combining high-throughput sequencing with in vivo experiments can reconstruct a global understanding of regulation, but also pave the way for discovery of compounds that target (ribo-)regulators to mitigate virulence and antibiotic resistance.
Highlights
The transcriptional architecture of bacterial genomes is far more complex than originally proposed
The canonical relationship between a bacterial operon and the mRNA transcript produced from the operon has become significantly more complex as numerous regulatory mechanisms that impact the stability, translational efficiency, and early termination rates for mRNA transcripts have been described
We find that the majority of multi-gene operons have alternative start and stop sites enabling condition specific regulation of genes within the same operon
Summary
The transcriptional architecture of bacterial genomes is far more complex than originally proposed. It seems likely that the bacterial transcriptional landscape, or the genomewide map of all possible transcriptional units, is shaped by an operon architecture that encodes many TSSs and TTSs within single operons, significantly increasing complexity with the objective of enabling diverse transcriptional outcomes [5,6]. In addition to the many protein activators and repressors that control transcription initiation, there are many non-coding RNAs (ncRNAs), including both small ncRNAs (sRNAs) and highly structured portions of mRNAs that play essential roles as regulatory elements controlling metabolism, stress-responses, and virulence [7,8,9]. Cis– acting mRNA structures, such as riboswitches, which interact with small molecules including metal ions, and protein ligands, and other regulatory sequences that are found in the long 3’ UTR of mRNAs, affect expression of their respective genes by regulating transcription attenuation or translation inhibition [12,13]. Several RNA regulators have been validated and associated with pathogenicity and
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