PPR proteins shed a new light on RNase P biology.

A fast growing number of studies identify pentatricopeptide repeat (PPR) proteins as major players in gene expression processes. Among them, a subset of PPR proteins called PRORP possesses RNase P activity in several eukaryotes, both in nuclei and organelles. RNase P is the endonucleolytic activity that removes 5' leader sequences from tRNA precursors and is thus essential for translation. Before the characterization of PRORP, RNase P enzymes were thought to occur universally as ribonucleoproteins, although some evidence implied that some eukaryotes or cellular compartments did not use RNA for RNase P activity. The characterization of PRORP reveals a two-domain enzyme, with an N-terminal domain containing multiple PPR motifs and assumed to achieve target specificity and a C-terminal domain holding catalytic activity. The nature of PRORP interactions with tRNAs suggests that ribonucleoprotein and protein-only RNase P enzymes share a similar substrate binding process

Processing of chloroplast ribosomal RNA transcripts in Euglena gracilis bacillaris

The ribosomal RNA operons ( rrn operons) of Euglena gracilis chloroplasts contain genes for (in order) 16S rRNA, tRNA Ile , tRNA Ala , 23S rRNA and 5S rRNA. Major sites of cleavage of the primary rrn transcript were identified by Northern blot hybridization and S1-mapping. The presumptive termini of all of the mature products have now been identified. During initial processing in the chloroplast, the primary transcript is cleaved between the two tRNAs and between the 23S and 5S rRNAs so as to separate the sequences found in the different mature rRNAs. Subsequently the tRNAs are separated from the rRNAs, further trimming provides the remaining proper ends, and the 3′-ends of the tRNAs are added.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46969/1/294_2004_Article_BF00419917.pd

Chloroplast ribonuclease P does not utilize the ribozyme-type pre-tRNA cleavage mechanism.

The transfer RNA 5' maturation enzyme RNase P has been characterized in Bacteria, Archaea, and Eukarya. The purified enzyme from all three kingdoms is a ribonucleoprotein containing an essential RNA subunit; indeed, the RNA subunit of bacterial RNase P RNA is the sole catalytic component. In contrast, the RNase P activity isolated from spinach chloroplasts lacks an RNA component and appears to function as a catalytic protein. Nonetheless, the chloroplast enzyme recognizes a pre-tRNA substrate for E. coli RNase P and cleaves it as efficiently and precisely as does the bacterial enzyme. To ascertain whether there are differences in catalytic mechanism between an all-RNA and an all-protein RNase P, we took advantage of the fact that phosphodiester bond selection and hydrolysis by the E. coli RNase P ribozyme is directed by a Mg2+ ion coordinated to the nonbridging pro-Rp oxygen of the scissile bond, and is blocked by sulfur replacement of this oxygen. We therefore tested the ability of the chloroplast enzyme to process a precursor tRNA containing this sulfur substitution. Partially purified RNase P from spinach chloroplasts can accurately and efficiently process phosphorothioate-substituted pre-tRNAs; cleavage occurs exclusively at the thio-containing scissile bond. The enzymatic throughput is fivefold slower, consistent with a general chemical effect of the phosphorothioate substitution rather than with a metal coordination deficiency. The chloroplast RNase P reaction mechanism therefore does not involve a catalytic Mg2+ bonded to the pro-Rp phosphate oxygen, and hence is distinct from the mechanism of the bacterial ribozyme RNase P

Evidence for an RNA-based catalytic mechanism in eukaryotic nuclear ribonuclease P.

Ribonuclease P is the enzyme responsible for removing the 5'-leader segment of precursor transfer RNAs in all organisms. All eukaryotic nuclear RNase Ps are ribonucleoproteins in which multiple protein components and a single RNA species are required for activity in vitro as well as in vivo. It is not known, however, which subunits participate directly in phosphodiester-bond hydrolysis. The RNA subunit of nuclear RNase P is evolutionarily related to its catalytically active bacterial counterpart, prompting speculation that in eukaryotes the RNA may be the catalytic component. In the bacterial RNase P reaction, Mg(II) is required to coordinate the nonbridging phosphodiester oxygen(s) of the scissile bond. As a consequence, bacterial RNase P cannot cleave pre-tRNA in which the pro-Rp nonbridging oxygen of the scissile bond is replaced by sulfur. In contrast, the RNase P reaction in plant chloroplasts is catalyzed by a protein enzyme whose mechanism does not involve Mg(II) coordinated by the pro-Rp oxygen. To determine whether the mechanism of nuclear RNase P resembles more closely an RNA- or a protein-catalyzed reaction, we analyzed the ability of Saccharomyces cerevisiae nuclear RNase P to cleave pre-tRNA containing a sulfur substitution of the pro-Rp oxygen at the cleavage site. Sulfur substitution at this position prohibits correct cleavage of pre-tRNA. Cleavage by eukaryotic RNase P thus depends on the presence of a thio-sensitive ligand to the pro-Rp oxygen of the scissile bond, and is consistent with a common, RNA-based mechanism for the bacterial and eukaryal enzymes