Verification of phylogenetic predictions in vivo and the importance of the tetraloop motif in a catalytic RNA

Abstract

M1 RNA, the catalytic subunit of Escherichia coli RNase P, forms a secondary structure that includes five sequence variants of the tetraloop motif. Site-directed mutagenesis of the five tetraloops of M1 RNA, and subsequent steady-state kinetic analysis in vitro, with different substrates in the presence and absence of the protein cofactor, reveal that (i) certain mutants exhibit defects that vary in a substratedependent manner, and that (ii) the protein cofactor can correct the mutant phenotypes in vitro, a phenomenon that is also substrate dependent. Thermal denaturation curves of tetraloop mutants that exhibit kinetic defects differ from those of wild-type M1 RNA. Although the data collected in vitro underscore the importance of the tetraloop motif to M1 RNA function and structure, three of the five tetraloops we examined in vivo are essential for the function of E. coli RNase P. The kinetic data in vitro are not in total agreement with previous phylogenetic predictions but the data in vivo are, as only mutants in those tetraloops proposed to be involved in tertiary interactions fail to complement in vivo. Therefore, the tetraloop motif is critical for the stabilization of the structure of M1 RNA and essential to RNase P function in the cell.Escherichia coli RNase P is a ribonucleoprotein enzyme composed of a single RNA (M1 RNA) and a single protein (C5 protein) subunit. Both subunits are essential for RNase P function in vivo. However, in vitro, M1 RNA alone can catalyze the hydrolysis of all the known RNA substrates of RNase P (1, 2), including precursor tRNAs (ptRNAs) and precursors to 4.5S and 10Sa RNAs (2).Phylogenetic studies of ribosomal RNA led to the identification of recurring secondary structural motifs such as that of the tetraloop (loops of 4 nts) at the turn of an RNA duplex that has, primarily, one of two sequence variations: GNRA or UNCG (where, N ϭ A, C, G or U; R ϭ A or G) (3). The presence of a tetraloop can confer on short model helices an added thermodynamic stability and therefore one role of a tetraloop might be to stabilize an RNA duplex in a functional RNA molecule (4-6). The tetraloop also could serve to stabilize the tertiary structure of an RNA molecule by making specific contact(s) with a distal site in the molecule (an intramolecular interaction) (7-9). Tetraloops also can form intermolecular interactions, for example, by mediating formation of an RNA-protein complex (10).M1 RNA has several tetraloops in its proposed secondary structure, as do many other RNAs with identifiable function. Specifically, of the eight loops at the turn of RNA helices in M1 RNA, five are tetraloops (one has the sequence UNCG and four have the sequence GNRA; see Fig. 1). It is not clear what function all the tetraloops of M1 RNA serve or whether they are necessary for RNase P function in vivo. The M1 RNA tetraloops could, in principle, (i) mediate interactions between M1 RNA and the C5 protein, (ii) mediate the binding of M1 RNA to some or all of its RNA substrates, or (iii) stabilize the conform...

A subunit of human nuclear RNase P has ATPase activity

“…coli RNase P has no ATPase activity. Previous reports (15,16) show that during precursor tRNA cleavage, some unwinding of the substrate occurs. By analogy, a similar unwinding of substrate must occur during cleavage by eukaryotic (human) RNase P. However, we have not yet been able to demonstrate directly any helicase activity associated with either E. coli or human RNase P with the substrate we used.…”

Eukaryotic Ribonuclease P: A Plurality of Ribonucleoprotein Enzymes

“…Studies of hairpins from ribosomal RNA and other sources have suggested that a tetraloop structure could increase the thermodynamic stability of an RNA duplex, or act as a docking site for intramolecular or intermolecular RNA-RNA interactions (124)(125)(126)(127)(128)(129)(130). In E. coli RNase P RNA (M1 RNA), a mutation in the L9 loop results in a reduction in K m and k cat of the holoenzyme-catalyzed reaction with pre-4.5S RNA as substrate (131), but little effect on pre-tRNA processing in vitro.…”

RNase P RNA from the Recently Evolved Plastid of Paulinella and from Algae

“…These tetraloops are important for stabilization of the tertiary structure of the RNA through long distance interactions between L9 and P1 [ 32 ], and between L14 and L18 with P8 [ 33 ] ( Figure 1 and Figure S2 ). These tertiary interactions have been shown to be functionally relevant in E. coli [ 34 ]. Therefore the absence of intramolecular stabilizing tertiary interactions could be a possible explanation for the catalytic deficit of plastid RPRs (below).…”