Marszalkowski-1-Comparative study on tertiary contacts and folding of RNase P RNAs from a 1 psychrophilic , a mesophilic / radiation-resistant and a thermophilic bacterium 2 3

29 30 In most bacterial type A RNase P RNAs (P RNAs), two major loop-helix tertiary contacts 31 (L8-P4 and L18-P8) help to orient the two independently folding Sand C-domains for 32 concerted recognition of precursor tRNA substrates. Here, we analyze the effects of 33 mutations in these tertiary contacts in P RNAs from three different species: (i) the 34 psychrophilic bacterium Pseudoalteromonas translucida (Ptr), (ii) the mesophilic 35 radiation-resistant bacterium Deinococcus radiodurans (Dra), and (iii) the thermophilic 36 bacterium Thermus thermophilus (Tth). We show by UV melting experiments that 37 simultaneous disruption of these two interdomain contacts has a stabilizing effect on all 38 three P RNAs. This can be inferred from reduced RNA unfolding at lower temperatures 39 and a more concerted unfolding at higher temperatures. Thus, when the two domains 40 tightly interact via the tertiary contacts, one domain facilitates structural transitions in the 41 other. P RNA mutants with disrupted interdomain contacts showed severe kinetic defects 42 that were most pronounced upon simultaneous disruption of the L8-P4 and L18-P8 43 contacts. At 37°C, the mildest effects were observed for the thermostable Tth RNA. A 44 third interdomain contact, L9-P1, makes only a minor contribution to P RNA tertiary 45 folding. Furthermore, D. radiodurans RNase P RNA forms an additional pseudoknot 46 structure between the P9 and P12 of its S-domain. This interaction was found to be 47 particularly crucial for RNase P holoenzyme activity at near-physiological Mg2+ 48 concentrations (2 mM). We further analyzed an exceptionally stable folding trap of the 49 G,C-rich Tth P RNA. 50 Cold Spring Harbor Laboratory Press on August 26, 2021 Published by rnajournal.cshlp.org Downloaded from