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. 1999 Sep 28;96(20):11200-5.
doi: 10.1073/pnas.96.20.11200.

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

D A Pomeranz Krummel  1 , S Altman
Affiliations

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

D A Pomeranz Krummel et al. Proc Natl Acad Sci U S A. .

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 substrate-dependent 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.

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Figures

Figure 1
Figure 1
Proposed Watson-Crick base pairing interactions, indicated by solid lines, and proposed tertiary interactions, indicated by dashed lines, of the secondary structure and three-dimensional computational model of E. coli M1 RNA (adapted from ref. 17). (A) Secondary structure of E. coli M1 RNA: broken line in bold demarcates two proposed independent folding domains of the RNA, domains 1 and 2 (19). Five tetraloops are indicated in color: L3 (pink), L9 (blue), L12 (yellow), L14 (green), and L18 (red) as are their proposed sites of intramolecular interaction (11, 12). (B) Three-dimensional computational model of E. coli M1 RNA (17). Five tetraloops are indicated in color: L3 (pink), L9 (blue), L12 (yellow), L14 (green), and L18 (red) as are their proposed sites of intramolecular interaction (11, 12). Nucleotide A89 (orange), and domains 1 (white) and 2 (cyan) are highlighted.
Figure 2
Figure 2
Enzymatic activity of wild-type (WT) M1 RNA and tetraloop mutants at 20 mM and 100 mM Mg2+. (A) Activity of wild-type and tetraloop mutants in 1× buffer A (see Materials and Methods) that contains 20 mM Mg2+. Reaction sampled at 4 and 8 min for wild type, as well as five tetraloop mutants. Control is an 8-min sample, under same conditions but in the absence of M1 RNA. (B) Activity of wild-type and tetraloop mutants M1 RNA in 1× buffer A that contains 100 mM Mg2+. Reactions sampled at 5 and 10 min. The precursor tRNATyr is indicated as pTyr; the product or mature tRNATyr is indicated as mTyr; the 5′ leader sequence is indicated as such.
Figure 3
Figure 3
Thermal denaturation profiles of wild-type M1 RNA and tetraloop mutants L14m and L18m. (A) Thermal denaturation profile of wild-type M1 RNA (black) and tetraloop mutants L14m (green) and L18m (red), expressed as normalized absorbance versus temperature. (B) First derivative of thermal denaturation profiles of wild-type M1 RNA (black) and tetraloop mutants L14m (green) and L18m (red). Structural transitions are noted at ≈57°C, ≈77°C, and ≈82°C. (Inset) Activity of wild-type M1 RNA as a function of temperature. To measure activity as a function of temperature, wild-type M1 RNA (5 nM) and pTyr (100 nM) were incubated at the desired temperature for 5 min, separately, before being mixed in the reaction mixture (see Materials and Methods).
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