Regulation of translation initiation by Mycobacterium tuberculosis non-coding RNAs observed at the single-molecule level and in real-time

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Mycobacterium tuberculosis (Mtb) infections are the leading cause of death resulting from a single pathogen. A major problem of treating Mtb is its unique life cycle with two different metabolic states: an active replicating disease state and a latent state. In this latter state, protein synthesis is globally down-regulated and therefore, bacteria are less susceptible to antibiotics. Non-coding RNAs (called small RNAs, sRNAs, in bacteria) may regulate entry into and maintenance of this dormant state and are also involved in active TB infection. sRNAs can act at several stages of gene expression and serve as global regulators of gene expression, but little is known about how Mtb sRNAs work at the molecular level to regulate Mtb gene expression. In this proposal, we will set up an in vitro multi-color single-molecule fluorescence platform for real-time tracking of co-transcriptional translation initiation regulated by Mtb sRNAs and validate these data with in vitro and in vivo functional assays. Tracking single mRNA molecules will allow us to directly see how transcription, co-transcriptional mRNA folding, interactions of the mRNA with sRNAs and translation initiation occur in real-time and are functionally coupled with each other. Our multi-color single-molecule platform and the resulting mechanistic findings will be applicable to a multitude of unexplored bacterial sRNAs. Our first goal with this project is to establish a single-molecule fluorescence microscopy platform for real-time tracking of Mtb co-transcriptional translation initiation. To accomplish this, we will 1) establish an assay for monitoring transcription elongation using Mtb RNA polymerase in real-time, 2) reconstitute and label in vitro Mtb 30S ribosomal subunits and 3) develop an integrated assay for simultaneous real-time monitoring of transcription elongation, mRNA folding and translation initiation to understand how nascent mRNA structure affects translation initiation dynamics. The second goal of this project is to investigate the molecular mechanisms of translation initiation regulation by the Mtb 6C sRNA as a model system. Here we will 1) study how the binding dynamics of the 6C sRNA regulate translation initiation efficiency of different mRNA targets and 2) investigate how other factors affect 6C sRNA function (e.g. RNA chaperones, antisense oligos).In summary, this single-molecule platform, the established methodologies and developed concepts will allow further studies of the so far little explored context of co-transcriptional mRNA structure formation and how dynamic RNA structure incorporates cellular signals (sRNAs, metabolites, environmental conditions) to convert them to a specific cellular output (e.g. successful translation initiation). RNA is dynamic and heterogeneous and therefore difficult to study and this proposal will deliver methodologies to tackle these challenges.