Protein-precursor tRNA contact leads to sequence-specific recognition of 5' leaders by bacterial ribonuclease P

Abstract

Bacterial ribonuclease P (RNase P) catalyzes the cleavage of 5' leader sequences from precursor tRNAs (pre-tRNAs). Previously, all known substrate nucleotide specificities in this system are derived from RNA-RNA interactions with the RNase P RNA subunit. Here, we demonstrate that pre-tRNA binding affinities for Bacillus subtilis and Escherichia coli RNase P are enhanced by sequence-specific contacts between the fourth pre-tRNA nucleotide on the 5' side of the cleavage site (N(-4)) and the RNase P protein (P protein) subunit. B. subtilis RNase P has a higher affinity for pre-tRNA with adenosine at N(-4), and this binding preference is amplified at physiological divalent ion concentrations. Measurements of pre-tRNA-containing adenosine analogs at N(-4) indicate that specificity arises from a combination of hydrogen bonding to the N6 exocyclic amine of adenosine and steric exclusion of the N2 amine of guanosine. Mutagenesis of B. subtilis P protein indicates that F20 and Y34 contribute to selectivity at N(-4). The hydroxyl group of Y34 enhances selectivity, likely by forming a hydrogen bond with the N(-4) nucleotide. The sequence preference of E. coli RNase P is diminished, showing a weak preference for adenosine and cytosine at N(-4), consistent with the substitution of Leu for Y34 in the E. coli P protein. This is the first identification of a sequence-specific contact between P protein and pre-tRNA that contributes to molecular recognition of RNase P. Additionally, sequence analyses reveal that a greater-than-expected fraction of pre-tRNAs from both E. coli and B. subtilis contains a nucleotide at N(-4) that enhances RNase P affinity. This observation suggests that specificity at N(-4) contributes to substrate recognition in vivo. Furthermore, bioinformatic analyses suggest that sequence-specific contacts between the protein subunit and the leader sequences of pre-tRNAs may be common in bacterial RNase P and may lead to species-specific substrate recognition.

Figure 1. Interaction of the 5’ leader of pre-tRNA with B. subtilis P protein in RNase P

(A) Modeled structure of the interface between the pre-tRNA leader and P protein based on affinity cleavage , showing the backbone of PRNA (blue), P protein (red), the mature tRNA domain of the substrate (brown), and the pre-tRNA 5’ leader (black). The nucleotide at pre-tRNA position N(−4) is shown in green; P protein regions are labeled. (B) B. subtilis P protein crystal structure showing sites where single cysteine mutations alter the value of K_{D, obs} for pre-tRNA^Asp. The side chain color reflects the magnitude of the effect of the mutation on the value of K_{D, obs}: yellow, ≤ 3-fold increase; blue, 3- to 25-fold increase; and green, > 25-fold increase. (C) The effect of alanine mutations on the binding selectivity for A(−4) relative G(−4) mapped onto the structure of the RNase P protein. Alanine mutations that do not alter the binding specificity ratio are highlighted in yellow and those that abolish the sequence preference at N(−4) are colored in green.

Figure 2. Dependence of pre-tRNA binding affinity on both the nucleotide at N(−4) and the calcium concentration

Representative binding isotherms for RNase P binding to pre-tRNA^Asp with varying sequence at N(−4) at 2 mM and 3.5 mM [Ca²⁺]_f. Fraction substrate bound was measured using centrifuge gel-filtration chromatography in 50 mM MES, 50 mM Tris, pH 6.0, 37 °C, with KCl concentrations were adjusted to maintain ionic strength at 410 mM. Data in A and B for A(−4) (filled circles; solid line), U(−4) (open circles; solid line), C(−4) (open squares; dashed line) and G(−4) (filled squares, dashed line) are fit with a single binding isotherm. C K_D,obs values for A(−4) (open circles) and G(−4) (filled circles) substrates as a function of [Ca²⁺]_f. Error bars represent the standard deviation of at least three independent trials. The solid line is the fit of Equation 1 to the G(−4) data; a dashed line shows the best linear fit to the A(−4) data. Inset: Hill plot of data for G(−4) between 2 and 5 mM [Ca²⁺]_f.

Figure 3. Binding preferences at N(−4) of pre-tRNA depend on Tyr34 in the P protein subunit

Isotherms for binding of B. subtilis RNase P to N(−4) pre-tRNA^Asp were measured as described in the legend of Figure 2 at 2 mM [Ca²⁺]_f for wild-type RNase P (closed symbols) and the Y34F mutant of RNase P (open symbols) with A(−4) pre-tRNA (circle) and G(−4) pre-tRNA (square).

Figure 4. Sequence preferences in genomic pre-tRNA Leaders

Sequence logos showing information content of 5’ leader in B. subtilis (A) and E. coli (B) pre-tRNA genes. Total bar height reflects increased information content relative to the background nucleotide content of the genome in the region of tRNA genes (See Materials and Methods). Error bars represent one standard deviation of the total bar height . C Percentage of 161 species examined with statistically significant (p ≤ 0.05) nucleotide enrichments at the indicated 5’ leader positions.

Figure 5. Distribution of Nucleotide Preferences at N(−4)

Nucleotide preferences at N(−4) for 20 representative bacterial species are shown. Bars show fractional composition of U (black), A (grey), G (white) and C (cross hatched). All species shown have statistically significant nucleotide enrichments (p < 0.05) preference at N(−4), and are arranged by overall A/U content at N(−4).