Crystal Structure of the tRNA Processing Enzyme RNase PH from Aquifex aeolicus

Abstract

RNase PH is one of the exoribonucleases that catalyze the 3 end processing of tRNA in bacteria. RNase PH removes nucleotides following the CCA sequence of tRNA precursors by phosphorolysis and generates mature tRNAs with amino acid acceptor activity. In this study, we determined the crystal structure of Aquifex aeolicus RNase PH bound with a phosphate, a co-substrate, in the active site at 2.3-Å resolution. RNase PH has the typical ␣/␤ fold, which forms a hexameric ring structure as a trimer of dimers. This ring structure resembles that of the polynucleotide phosphorylase core domain homotrimer, another phosphorolytic exoribonuclease. Four amino acid residues, Arg-86, Gly-124, Thr-125, and Arg-126, of RNase PH are involved in the phosphate-binding site. Mutational analyses of these residues showed their importance in the phosphorolysis reaction. A docking model with the tRNA acceptor stem suggests how RNase PH accommodates substrate RNAs.The processing of tRNA precursors into mature tRNAs involves several steps, including the removal of the 5Ј and 3Ј extension sequences (1-4). This removal of the 5Ј and 3Ј extension sequences is necessary for producing the CCA sequence, which is essential for tRNA aminoacylation and subsequent protein synthesis (2-4). The 5Ј extension is removed, in a single step reaction, by the ribonucleoprotein RNase P, which is conserved in all organisms (5). On the other hand, the removal of the 3Ј extension is a more complex process. In archaea and eukaryotes, the 3Ј extension sequence is removed by RNase Z in a single step reaction (6, 7), whereas in bacteria a number of RNases perform this function in multistep reactions (2-4). In Escherichia coli, the proposed 3Ј processing pathway involves the following steps (2-4). First, an endonucleolytic cleavage occurs downstream of the CCA sequence. RNases E and III have been identified as participating in this first step. Then the endonucleolytic reaction is followed by the 5Ј-terminal processing by RNase P, if the 3Ј trailer sequence of the tRNA precursor is short. However, if the tRNA precursor has a long 3Ј trailer sequence, an exonucleolytic reaction removes some nucleotides from the 3Ј end before RNase P processes the 5Ј end. This exonucleolytic reaction involves RNases PH, T, BN, D, II, and polynucleotide phosphorylase. Finally, these exonucleases remove all of the nucleotides downstream of the CCA sequence; RNase PH and RNase T are usually most effective (8).In this final trimming, RNase PH catalyzes a 3Ј exonucleolytic, phosphorolytic reaction, which needs a phosphate as the co-substrate, and produces a nucleoside diphosphate (9, 10). RNase PH was shown to catalyze the reverse reaction in vitro,

The Phosphorolytic Exoribonucleases Polynucleotide Phosphorylase and RNase PH Stabilize sRNAs and Facilitate Regulation of Their mRNA Targets

“…PNPase possesses KH and S1 domains that assist in binding RNA and guiding it to one of three active sites present in the PNPase trimer (21,39). In contrast, RNase PH forms a simple toroid structure that is capable of binding RNA on both faces of its hexamer (22). These structural differences may have important consequences on how each protein is able to engage and interact with RNA or other proteins.…”

“…These two RNases share significant core structural features (21,22), are widely conserved throughout bacteria (23), and play important roles in processing the 3= ends of tRNAs and other small structured RNAs (24)(25)(26). Although E. coli tolerates single deletions of either protein, a double deletion results in slower growth and cold sensitivity (27).…”

Structural and biochemical characterization of CRN-5 and Rrp46: An exosome component participating in apoptotic DNA degradation

“…The CRN-5 structure is the first homodimeric structure observed in the RNase PH family proteins, the rest of which all crystallized in trimeric or hexameric forms with six RNase PH domains forming a ring-like structure, including the human exosome , the archaeal exosome (Buttner et al 2005;Navarro et al 2008), bacterial PNPase (Symmons et al 2000;Shi et al 2008), and RNase PH (Ishii et al 2003;Harlow et al 2004). Superposition of the CRN-5 dimer over hRrp46-hRrp43 gave a RMSD of 3.05 Å over 273 C a atoms, whereas superposition of the CRN-5 dimer onto the archaeal Rrp41-Rrp42 dimer (PDB accession code: 2JEA) gave a RMSD of 3.02 Å over 279 C a atoms.…”

The PNPase, exosome and RNA helicases as the building components of evolutionarily-conserved RNA degradation machines

“…Similarities in the structure of the RNase PH and 3¢ fi 5¢ RNA degradation machines: the PNPase and the exosome. The structure of the RNase PH [22,23], the bacterial PNPase [27], archaeal [29,30] and eukaryotic [31] exosomes, as well as the predicted structure of the chloroplast PNPase [24], are shown in order to compare the ring shape of these complexes. The molecular surfaces of these structures are represented in the same view and colored as in Figure 2.…”

“…RNase PH homologs are small single domain proteins that are distributed among all three primary kingdoms. In archaea and eukaryotes they form the core of the exosome complexes ( Figure 2); in bacteria, six RNase PH polypeptides form a homohexameric structure [22,23] that is similar to that of the PNPase and the exosome (see below; Figures 2 and 3).…”