Mette Kongstad:
Bacterial growth physiology. With focus on the tRNA-linked-repeats and tRNA regulation during starvation

Date: 30-04-2016    Supervisor: Michael Askvad Sørensen & Sine Lo Svenningsen

Transfer RNA (tRNA) mediates the translation of the code, transiently stored in the messenger RNA (mRNA), to the final protein. The entity of tRNA has for decades been assumed to be stable for hours in any circumstance, but my supervisor Michael A. Sørensen noticed during his work with charging level of tRNA that the tRNA level was seemingly reduced much faster than observed before, after the induction of amino acid starvation. Therefore, we developed a method for testing this observation. It turned out that tRNA degrades to ~75% of the initial level within 40 minutes after induction of amino acid starvation. Our next task was to explore the mechanism behind the starvation-induced downregulation of tRNA, and the obvious candidates as RelA, SpoT, and the E.coli toxin-anti toxin system (TA) were tested with no effect. Interestingly, the lifetime of tRNA was prolonged in an isogenic Δhfq after the induction of amino acid starvation. Indeed, the RNA chaperone Hfq is known to facilitate a pairing of small RNA (sRNA) with their target mRNA, affecting gene expression. Thus, our attention was drawn towards sRNA and other regulatory RNAs. The tRNA-linked-repeats (TLR) acting as regulatory RNA (regRNA) in collaboration with Hfq seemed to be good candidates for starvation-induced down regulation of tRNA. This is because TLRs are small intergenic repeats located in operons of tRNA and rRNA that share 18-19 nt of homology with the RNA they are located next to with yet an unknown function. It turned out that the TLRs were not involved in starvation-induced down regulation of tRNA. As such, my project was subdivided into the following segments:

1 Part: starvation-induced regulation of tRNA

2 Part: TLRs

The TLRs were not involved in tRNA regulation. Instead, three TLRs showed themselves to be enriched at Hfq, and these TLRs displayed increased stability upon Hfq interaction. From this and other experiments, we propose that the three TLRs transcribed from the rrnA, rrnB, and rrnD operons act as regRNAs, assisted by Hfq.