The dawn of dominance by the mature domain in tRNA splicing

Abstract

The relationship between enzyme architecture and substrate specificity among archaeal pre-tRNA splicing endonucleases has been investigated more deeply, by using biochemical assays and model building. The enzyme from Archeoglobus fulgidus (AF) is particularly interesting: it cleaves the bulge-helix-bulge target without requiring the mature tRNA domain, but, when the target is a bulge-helix-loop, the mature domain is required. A model of AF based on its electrostatic potential shows three polar patches interacting with the pre-tRNA substrate. A simple deletion mutant of the AF endonuclease lacking two of the three polar patches no longer cleaves the bulge-helix-loop substrate with or without the mature domain. This single deletion shows a possible path for the evolution of eukaryal splicing endonucleases from the archaeal enzyme.

Fig. 1.

Substrates for the in vitro cleavage assays. (A) PretRNA^BHB consists of two regions derived from yeast pre-tRNA^PHE (nucleotides 1–31 and 38–76), joined by a 25-nt insert that corresponds to the BHB motif of archaeal tRNA^Trp. (B) Mini-BHB is the BHB motif of pre-tRNA^BHB. (C) PretRNA^BHL corresponds to pre-tRNA^Tyr from Caenorhabditis elegans. The synthetic substrate presents a residue change at the 5′ terminus (C to G) required for T7 transcription and a corresponding change (G to C) in the complementary strand. (D) Mini-BHL is the BHL motif of pre-tRNA^BHL. The base substitutions in the mini substrates were introduced to optimize T7 RNA polymerase transcription (5).

Fig. 2.

In vitro cleavage of BHB- and BHL-containing substrates. (A) The mini–BHB substrate was incubated with three different enzymes. (B) The mini–BHL substrate was incubated with three different enzymes. (C) Pre-tRNA^BHB was incubated with three different enzymes. (D) PretRNA^BHL was incubated with three different enzymes. The conditions of the reactions have been reported (15). The cleavage products were analyzed by electrophoresis on 10% polyacrylamide gel containing 29:1 monomer to bis and 8 M urea, followed by autoradiography. The identification of the reaction products is indicated. Lane 1 contains the control (C, no enzyme added). Lanes 2–4 show the products after incubation with the endonucleases from MJ, AF, and SS, respectively. The 2/3 molecules are produced by single cleavage.

Fig. 3.

Electrostatic surface potentials of MJ (A) and AF (B) endonucleases. Positively charged regions are shown in blue, the neutral regions are shown in white, and the negatively charged regions are shown in red. The green circle encompasses the catalytic site, whereas the cyan circles enclose the two putative sites recognizing the tRNA mature domain. The figure was generated with PyMOL (http://pymol.sourceforge.net), and the electrostatic properties were generated by using the APBS (Adaptive Poisson-Boltzman Solver) software package (16).

Fig. 4.

Structure of ΔAF enzyme and model of the interaction with its substrate. (A) Cartoon representation of the parts deleted from AF endonuclease (gray). The arrows indicate the new N termini of the subunits. The BHB substrate is shown in green. (B) Electrostatic surface potential of ΔAF. Positively charged regions are shown in blue, the neutral regions are shown in white, and the negatively charged regions are shown in red. The pre-tRNA model is shown in cyan.

Fig. 5.

In vitro cleavage of BHB and BHL containing substrates by the ΔAF enzyme. (A) The mini-BHB substrate was incubated with the AF and ΔAF enzymes. (B) The mini-BHL substrate was incubated with the AF and ΔAF enzymes. (C) The pre-tRNA^BHB substrate was incubated with the AF and ΔAF enzymes. (D) The pre-tRNA^BHL substrate was incubated with the AF and ΔAF enzymes. The conditions of the reactions have been reported (12). The cleavage products were analyzed by electrophoresis on 10% polyacrylamide gel containing 29:1 monomer to bis and 8 M urea, followed by autoradiography. The identification of the reaction products is indicated. Lane 1 contains the control (C, no enzyme added). Lanes 2 and 3 show the products after incubation with the endonucleases from AF and ΔAF, respectively. The 2/3 molecules are produced by single cleavage.

Fig. 6.

Model of the interactions between the AF enzyme and the substrates. Electrostatic surface potential of AF bound either to two BHB RNA molecules (shown in green) obtained from the crystallographic structure (11) (A) or to a model of pre-tRNA (shown in cyan) (B).

Fig. 7.

Model of a chimeric enzyme presenting two CAFs, one NAF, and the human Sen15. (A) Representation of the model chimeric enzyme. The archaeal parts are shown in gray, and the human part is shown in red. (B) Electrostatic surface potential of the model chimeric enzyme. Positively charged regions are shown in blue, neutral regions are shown in white, and negatively charged regions are shown in red. (C) Primary sequences and secondary structures of the NAF and of the human Sen15. Secondary structure elements, as determined by the crystallographic structure of AF (19) and the NMR structure of Sen15 (24), are shown in black and red, respectively. The “s” and β indicate β-strands, and “h” and α indicate α-helices. Black columns indicate residues that are conserved; gray columns indicate residues that are similar.