Kinetic analysis of 3'-5' nucleotide addition catalyzed by eukaryotic tRNA(His) guanylyltransferase

The tRNA(His) guanylyltransferase (Thg1) catalyzes the incorporation of a single guanosine residue at the -1 position (G(-1)) of tRNA(His), using an unusual 3'-5' nucleotidyl transfer reaction. Thg1 and Thg1 orthologs known as Thg1-like proteins (TLPs), which catalyze tRNA repair and editing, are the only known enzymes that add nucleotides in the 3'-5' direction. Thg1 enzymes share no identifiable sequence similarity with any other known enzyme family that could be used to suggest the mechanism for catalysis of the unusual 3'-5' addition reaction. The high-resolution crystal structure of human Thg1 revealed remarkable structural similarity between canonical DNA/RNA polymerases and eukaryotic Thg1; nevertheless, questions regarding the molecular mechanism of 3'-5' nucleotide addition remain. Here, we use transient kinetics to measure the pseudo-first-order forward rate constants for the three steps of the G(-1) addition reaction catalyzed by yeast Thg1: adenylylation of the 5' end of the tRNA (k(aden)), nucleotidyl transfer (k(ntrans)), and removal of pyrophosphate from the G(-1)-containing tRNA (k(ppase)). This kinetic framework, in conjunction with the crystal structure of nucleotide-bound Thg1, suggests a likely role for two-metal ion chemistry in all three chemical steps of the G(-1) addition reaction. Furthermore, we have identified additional residues (K44 and N161) involved in adenylylation and three positively charged residues (R27, K96, and R133) that participate primarily in the nucleotidyl transfer step of the reaction. These data provide a foundation for understanding the mechanism of 3'-5' nucleotide addition in tRNA(His) maturation.     

The final cut. The importance of tRNA 3'-processing

To generate functional tRNA molecules, precursor RNAs must undergo several processing steps. While the enzyme that generates the mature tRNA 5′-end, RNase P, has been thoroughly investigated, the 3′-processing activity is, despite its importance, less understood. While nothing is known about tRNA 3′-processing in archaea, the phenomenon has been analysed in detail in bacteria and is known to be a multistep process involving several enzymes, including both exo- and endonucleases. tRNA 3′-end processing in the eukaryotic nucleus seems to be either exonucleolytic or endonucleolytic, depending on the organism analysed, whereas in organelles, 3′-end maturation occurs via a single endonucleolytic cut. An interesting feature of organellar tRNA 3′-processing is the occurrence of overlapping tRNA genes in metazoan mitochondria, which presents a unique challenge for the mitochondrial tRNA maturation enzymes, since it requires not only the removal but also the addition of nucleotides by an editing reaction.     

Sequence motifs that distinguish ATP(CTP):tRNA nucleotidyl transferases from eubacterial poly(A) polymerases

ATP(CTP):tRNA nucleotidyl transferases, tRNA maturing enzymes found in all organisms, and eubacterial poly(A) polymerases, enzymes involved in mRNA degradation, are so similar that until now their biochemical functions could not be distinguished by their amino acid sequence. BLAST searches and analysis with the program "Sequence Space" for the prediction of functional residues revealed sequence motifs which define these two protein families. One of the poly(A) polymerase defining motifs specifies a structure that we propose to function in binding the 3' terminus of the RNA substrate. Similar motifs are found in other homopolyribonucleotidyl transferases. Phylogenetic classification of nucleotidyl tranferases from sequenced genomes reveals that eubacterial poly(A) polymerases have evolved relatively recently and are found only in a small group of bacteria and surprisingly also in plants, where they may function in organelles.     

Identification of multiple RNases in Xenopus laevis oocytes and their possible role in tRNA processing.

A survey of RNases in Xenopus laevis oocytes has been carried out to identify potential tRNA-processing enzymes in this system. Using a variety of specific and nonspecific substrates, we have shown that oocytes contain multiple RNases with various specificities. Three activities that could cleave the extraneous residues from the artificial tRNA precursor, tRNA-C-[14C]U-C, to generate a substrate for -C-C-A addition by tRNA nucleotidyltransferase were identified. One of these was a cytoplasmic exonuclease which generated predominantly tRNA-C, whereas the other two were nuclear endonucleases which cleaved the precursor to generate tRNA-N. The possible involvement of these activities in 3' tRNA processing in oocytes is discussed.