Mechanistic and structural analyses of different non-coding RNAs in bacteria Mechanistische und strukturelle Untersuchungen verschiedener nicht kodierender RNAs in Bakterien

RNase P RNA and 6S RNA are two non coding RNAs found in essentially all (RNase P RNA) or most (6S RNA) known bacteria. The tRNA maturation function of RNase P is highly essential but until now the catalytic mechanism is not fully understood. We started to investigate the two metal-ion-based mechanism via dynamical NMR measurements. For this purpose, we used a shortened variant of the Escherichia coli (E. coli) RNase P RNA (~370 nt) which lacks most of the specificity domain (E. coli RNase P RNA catalytic domain termed Ecat) to minimize the amount of NMR signals. Furthermore, we designed several hairpin-like minimal substrates containing a 5’ leader to determine nucleotides (substrate and Ecat) that are involved in the catalytically process. Due to the fact, that Ecat lost the function to cleave a substrate alone, the E. coli RNase P protein (C5 protein) was essential for the reaction. Here i) the homogeneity of the Ecat variant ii) the stability of C5-protein at room temperature and iii) the cleavability of the minimal substrates was tested. Further investigations on the usability of the minimal substrates for NMR measurements were performed. 6S RNA is a bacterial small non-coding RNA with a length of approximately 200 nt. First described in E. coli it was later detected in all branches of the bacterial kingdom [Barrick et al., 2005]. Among them the well-known model organisms E. coli and B. subtilis (Bacillus subtilis), but also extremophilic organisms such as the Aquificales bacteria were predicted to harbor a 6S RNA. The function of 6S RNA remained elusive for decades. Nowadays it is known that 6S RNA is a transcriptional regulator, binding RNA polymerases (RNAP) containing the housekeeping sigma factor to inhibit gene expression upon transition into stationary growth phase [Wassarman and Storz, 2000; Beckmann, Hoch et al., 2012]. Although 6S RNA is thought to compete with 70 (E. coli) or A (B. subtilis)- dependent promoters for binding to A/70 RNAP by mimicking an open DNA promoter structure also promoters regulated by other sigma factors were found to be affected by a 6S RNA knockout [Wassarman, 2007]. When bound to RNAP 6S RNA is used as a template for the synthesis of short 'product' RNAs (pRNAs). While shorter pRNAs (~ 8-mers) dissociate from the complex, transcription of pRNAs exceeding a certain minimal length leads to a persistent rearrangement of the 6S RNA structure which results in the dissociation of 6S RNA:RNAP complexes [Beckmann and Hoch et al., 2012; Steuten et al., 2014]. A 6S RNA homolog was also identified in an experimental RNomics study based on a cDNA library derived from small RNAs expressed in the hyperthermophilic bacterium Aquifex aeolicus [Willkomm et al., 2005]. A. aeolicus 6S RNA is predicted to form a canonical rod-shaped secondary structure with a central bulge region. The central bulge is flanked by two stem structures: i) the terminal or closing stem formed by the bases located at the 5’ and 3’ end of 6S RNA and ii) the internal stem arranged in a hairpin like structure. Comprising only 163 nt and a G/C- ratio of ~ 61% this 6S RNA exhibits the most stable structure among all known 6S RNAs. In this work the secondary structure of A. aeolicus 6S RNA and its structural change after pRNA transcription were investigated by NMR. NMR studies were performed on free 6S RNA as well as 6S RNA:pRNA hybrids, using a putative pRNA 15 mer previously found by deep sequencing analysis. Due to the fact that an maximal RNA size for NMR measurements is approx. 80 nt, the A. aeolicus 6S RNA was shortened either at the terminal stem (132-nt 6S RNA variant) or at both (terminal and internal) stem regions (85-nt 6S RNA variant). By analyzing these two shortened 6S RNA variants via NMR, we were able to verify formation of several predicted structural stem elements within the terminal and the internal stem, as well as predicted structural transitions upon pRNA binding. Additionally, the secondary structure of A. aeolicus 6S RNA was investigated by enzymatic and chemical structure probing to complement the NMR data in order to obtain a more fine-grained model of the RNA’s solution structure. We could finally clarify that the free 6S RNA is formed as expected, whereas the solved structure of the 6S RNA:pRNA hybrid shows intense divergence to the prediction. First of all, we could show that the terminal stem remains solidly paired even after the pRNA induced rearrangement of the 6S RNA structure. Moreover, hairpin formation in the 3´ central bulge (CB), which is known e.g. from B. subtilis 6S RNA, could not be detected. In line with the prediction we were able to prove the pRNA mediated formation of the CB collapse helix. Besides the structural investigation of 6S RNA we were interested in mechanistic aspects. To determine if A. aeolicus 6S RNA shares hallmark features with canonical 6S RNAs we employed the RNAP from B. subtilis which is well investigated in terms of 6S RNA binding and pRNA transcription [Beckmann et al., 2011; Beckmann, Hoch et al., 2012; Burenina et al., 2014]. Using B. subtilis RNAP for in vitro transcription, the 6S RNA of A. aeolicus was demonstrated to serve as a template for pRNA transcription and to undergo the structural rearrangement upon pRNA hybridization comparable to already known 6S RNAs of E. coli and B. subtilis. However, inhibiting formation of the central bulge collapse helix leads to inefficient 6S RNA:pRNA release from the RNAP complex, in A. aeolicus as well as in B. subtilis. This observation suggests that an efficient release of 6S RNA:pRNA hybrids from the complex with RNAP can only be induced by pRNAs whose transcription is initiated at their canonical position in 5’ CB.