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Biology subjects

Lilic, M.

Publications and source records attributed to Lilic, M..

4 recordsLinked to original sources

Incomplete transcripts dominate the Mycobacterium tuberculosis transcriptome

Mycobacterium tuberculosis (Mtb) is a bacterial pathogen that causes tuberculosis, an infectious disease that inflicts major health and economic costs around the world1. Mtb encounters a diversity of environments during its lifecycle, and responds to these changing environments by reprogramming its transcriptional output2. However, the transcriptomic features of Mtb remain poorly characterized. In this work, we comprehensively profile the Mtb transcriptome using the SEnd-seq method that simultaneously captures the 5 and 3 ends of RNA3. Surprisingly, we find that the RNA coverage for most of the Mtb transcription units display a gradual drop-off within a 200-500 nucleotide window downstream of the transcription start site, yielding a massive number of incomplete transcripts with heterogeneous 3 ends. We further show that the accumulation of these short RNAs is mainly due to the intrinsically low processivity of the Mtb transcription machinery rather than trans-acting factors such as Rho. Finally, we demonstrate that transcription-translation coupling plays a critical role in generating full-length protein-coding transcripts in Mtb. In sum, our results depict a mycobacterial transcriptome that is dominated by incomplete RNA products, suggesting a distinctive set of transcriptional regulatory mechanisms that could be exploited for new therapeutics.

microbiology↗

Structural and functional basis of the universal transcription factor NusG pro-pausing activity in Mycobacterium tuberculosis

Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger elemental pausing by stabilizing a half-translocated (RNA-not-DNA) state and by promoting RNAP swiveling that other factors can enhance. The universal transcription factor NusG (Spt5 in eukaryotes and archaea) N-terminal domain (NGN) modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs (e.g., Bacillus subtilis) enhance some pauses whereas anti-pausing NusGs (e.g., Escherichia coli) suppress some pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). Using biochemistry and cryo-electron microscopy, we show that MtbNusG is a pro-pausing NusG that captures paused, swiveled RNAP by contacts to the RNAP protrusion and to a nontemplate strand-DNA wedge inserted between the NGN and the RNAP gate loop. On the other hand, we find that anti-pausing E. coli NGN contacts the RNAP gate loop to inhibit swiveling and pausing of MtbRNAP. Using CRISPR-mediated mycobacterial genetics, we show that a pro-pausing NGN is required to support robust mycobacterial growth. Our results define an essential function of NusG in mycobacteria and the structural basis of pro-vs. anti-pausing NusG activity with broad implications for NusG function in all domains of life.

molecular biology↗

Immunodominant surface epitopes power immune evasion in the African trypanosome

The African trypanosome survives the immune response of its mammalian host by antigenic variation of its major surface antigen (the Variable Surface Glycoprotein, or VSG). Here we describe the antibody repertoires elicited by different VSGs. We show that the repertoires are highly restricted, and are directed predominantly to distinct epitopes on the surface of the VSGs. They are also highly discriminatory: minor alterations within these exposed epitopes confer antigenically-distinct properties to these VSGs and elicit different repertoires. We propose that the patterned and repetitive nature of the VSG coat focuses host immunity to a restricted set of immunodominant epitopes per VSG, eliciting a highly stereotyped response, minimizing cross reactivity between different VSGs and facilitating prolonged immune evasion through epitope variation.

immunology↗

Fidaxomicin jams M. tuberculosis RNA polymerase motions needed for initiation via RbpA contacts

Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against Mycobacterium tuberculosis RNAP in vitro, but clinical use of Fdx is limited to treating Clostridium difficile intestinal infections due to poor absorption. To enable structure-guided optimization of Fdx to treat tuberculosis, we report the 3.4 [A] cryo-electron microscopy structure of a complete M. tuberculosis RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin and explains its strong effect on M. tuberculosis. We present additional structures that define conformational states of M. tuberculosis RNAP between the free apo-holenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on Fdx.

biophysics↗