A eubacterial origin for the human tRNA nucleotidyltransferase?

tRNA CCA-termini are generated and maintained by tRNA nucleotidyltransferases. Together with poly(A) polymerases and other enzymes they belong to the nucleotidyltransferase superfamily. However, sequence alignments within this family do not allow to distinguish between CCA-adding enzymes and poly(A) polymerases. Furthermore, due to the lack of sequence information about animal CCA-adding enzymes, identification of corresponding animal genes was not possible so far. Therefore, we looked for the human homolog using the baker's yeast tRNA nucleotidyltransferase as a query sequence in a BLAST search. This revealed that the human gene transcript CGI-47, (\#AF151805) deposited in GenBank is likely to encode such an enzyme. To identify the nature of this protein, the cDNA of the transcript was cloned and the recombinant protein biochemically characterized, indicating that CGI-47 encodes a bona fide CCA-adding enzyme and not a poly(A) polymerase. This confirmed animal CCA-adding enzyme allowed us to identify putative homologs from other animals. Calculation of a neighbor-joining tree, using an alignment of several CCA-adding enzymes, revealed that the animal enzymes resemble more eubacterial ones than eukaryotic plant and fungal tRNA nucleotidyltransferases, suggesting that the animal nuclear cca genes might have been derived from the endosymbiotic progenitor of mitochondria and are therefore of eubacterial origin

3' processing of tRNA precursors in ribonuclease-deficient Escherichia coli. Development and characterization of an in vitro processing system and evidence for a phosphate requirement

In order to determine the mechanism and enzyme(s) responsible for 3' processing of tRNA precursors, we have developed an in vitro processing system that uses as substrates two SP6 RNA polymerase-generated transcripts of the gene for tRNA(Tyrsu3)+ that contain 49 extra 5'-nucleotides and either 5 or 25 extra 3'-nucleotides. A high speed supernatant fraction from an Escherichia coli strain deficient in five ribonucleases was found to accurately process both tRNA precursors in this system to the size of mature tRNA(Tyr). Final 3' end processing of each precursor occurs in an exonucleolytic manner to generate the correct 3' terminus; however, a prior endonucleolytic cleavage also is observed in processing of the longer precursor. The system requires Mg2+ and works optimally at about 50 mM KCl and pH 8-9. Dialysis of the supernatant fraction leads to loss of processing activity but can be restored to normal by the addition of inorganic phosphate or arsenate. Furthermore, nucleoside diphosphates are a product of the processing reaction. These data indicate that 3' processing in RNase-deficient extracts involves a phosphorolytic reaction. On the other hand, phosphate is not required for processing in RNase+ extracts, although it does aid in processing of the longer precursor. The usefulness of this in vitro system for studies of tRNA processing and the identity of the phosphate-requiring enzyme are discussed

Apparent involvement of ribonuclease D in the 3' processing of tRNA precursors.

Escherichia coli RNase D and RNase II have been purified to homogeneity and compared for their ability to remove extra nucleotides following the -C-C-A sequence in tRNA precursors. RNase D and RNase II are single-chain proteins with molecular weights of 38,000 and 78,000, respectively. Both enzymes require a divalent cation for activity on tRNA precursors, but, in addition, RNase II is stimulated by monovalent cations. RNase D specifically removes mononucleotide residues from a mixture of tRNA precursors to generate amino acid acceptor activity for essentially all amino acids. Although RNase II can also remove precursor-specific residues, no amino acid acceptor activity is recovered. Similarly, RNase D action on the E. coli tRNATyr precursor is limited, whereas RNase II causes extensive degradation. In contrast to the processive mode of hydrolysis by RNase II, RNase D removes nucleotides randomly and slows down greatly at the -C-C-A sequence, thereby allowing the tRNA to be aminoacylated and protected from further degradation. These results suggest that RNase D is the 3'-processing nuclease in vivo and that RNase II is a nonspecific degradative enzyme. The importance of RNA conformation for correct processing is also discussed

Escherichia coli RNase D. Purification and structural characterization of a putative processing nuclease

RNase D, a putative tRNa processing nuclease, has been purified about 1,000-fold from extracts of Escherichia coli to apparent homogeneity, as judged by acrylamide gel electrophoresis under nondenaturing and denaturing conditions and by gel electrofocusing. The purified enzyme is a single chain protein with a molecular weight of 40,000 and an isoelectric point of about 6.2. Spectral analysis indicated that RNase D is devoid of nucleic acid. Amino acid analysis suggested a low content of cysteine, and this was confirmed by the relative insensitivity of the enzyme to sulfhydryl group reagents. RNase D is sensitive to inactivation by elevated temperatures but can be protected by a variety of RNAs, including those which are not substrates for hydrolysis. The relation of RNase D to other known E. coli ribonucleases and to other previously identified processing activities, is discussed

Escherichia coli RNase D. Catalytic properties and substrate specificity

The catalytic properties of purified RNase D were examined. The enzyme requires a divalent cation for activity, and this requirement can be satisfied by Mg2+, MN2+, or Co2+. RNase D is most active at pH 9.1-9.5, but this optimum may reflect an effect on the substrate as well as the enzyme. A variety of RNAs were tested as substrates for RNase D. Alteration of the 3'-terminal base has no effect on the rate of hydrolysis, whereas modification of the 3'-terminal sugar has a major effect. tRNA terminating with a 3'-phosphate is completely inactive as a substrate. The rate of hydrolysis of intact tRNA is very slow compared to tRNAs containing extra residues or compared to tRNAs from which part of the -C-C-A sequence has been removed. Oxidation of the terminal sugar, reduction of the dialdehyde with borohydride, or removal of the terminal AMP from intact tRNa increase the activity of the substrate. Addition of a second -C-C-A sequence gives an active substrate indicating that the relative resistance of intact tRNA to RNase D hydrolysis is not due to the sequence per se but to the structural environment of the 3'-terminus. Studies of the mode of action of RNase D indicate that it is an exonuclease which initiates hydrolysis at the 3'-terminus and removes 5'-mononucleotides in a random fashion. The requirements of RNase D for interaction with nucleic acids and for hydrolysis of various RNAs and the relation of these properties to its possible role as a processing nuclease is discussed