A Phylogeny of Bacterial RNA Nucleotidyltransferases: Bacillus halodurans Contains Two tRNA Nucleotidyltransferases

Abstract

We have analyzed the distribution of RNA nucleotidyltransferases from the family that includes poly(A) polymerases (PAP) and tRNA nucleotidyltransferases (TNT) in 43 bacterial species. Genes of several bacterial species encode only one member of the nucleotidyltransferase superfamily (NTSF), and if that protein functions as a TNT, those organisms may not contain a poly(A) polymerase I like that of Escherichia coli. The genomes of several of the species examined encode more than one member of the nucleotidyltransferase superfamily. The function of some of those proteins is known, but in most cases no biochemical activity has been assigned to the NTSF. The NTSF protein sequences were used to construct an unrooted phylogenetic tree. To learn more about the function of the NTSFs in species whose genomes encode more than one, we have examined Bacillus halodurans. We have demonstrated that B. halodurans adds poly(A) tails to the 3 ends of RNAs in vivo. We have shown that the genes for both of the NTSFs encoded by the B. halodurans genome are transcribed in vivo. We have cloned, overexpressed, and purified the two NTSFs and have shown that neither functions as poly(A) polymerase in vitro. Rather, the two proteins function as tRNA nucleotidyltransferases, and our data suggest that, like some of the deep branching bacterial species previously studied by others, B. halodurans possesses separate CC-and A-adding tRNA nucleotidyltransferases. These observations raise the interesting question of the identity of the enzyme responsible for RNA polyadenylation in Bacillus.RNA nucleotidyltransferases play an important role in the maturation, processing, and degradation of RNAs. tRNA nucleotidyltransferases (TNT) and poly(A) polymerases (PAP) are members of a superfamily of RNA nucleotidyltransferases (13, 39). tRNA nucleotidyltransferases add CCA residues to the 3Ј ends of immature tRNAs and repair the 3Ј ends of damaged tRNAs (36). tRNA nucleotidyltransferases (TNTs) are found in bacteria, archaeae, and eukaryotes, suggesting conservation of structure and function of this class of enzymes over the course of evolution (39). Polyadenylation of RNA 3Ј ends, once thought to occur exclusively in eukaryotic cells, has now been shown definitively to occur in bacterial systems as well (7,25,30,31). Escherichia coli cells contain at least two enzymes capable of adding poly(A) tails to the 3Ј ends of RNA molecules. The major polyadenylating enzyme, poly(A) polymerase I (PAP I), was originally identified by virtue of its role in regulating plasmid copy number and is a product of the pcnB gene (6, 12). PAP I can utilize any of the three nucleoside triphosphates as substrates in vitro, but only A residues are added by the enzyme to RNA 3Ј ends in vivo (38). Messenger RNAs and 16S and 23S ribosomal RNAs are all polyadenylated in E. coli, and this modification is an important element of the RNA degradation pathway in bacterial systems (7,25,30,31). RNA 3Ј ends are generated in E. coli via endonucleolytic cleavage by RNase E and RNase III, thes...

Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases

“…10B). Thus, as previously observed (24), poly(A) polymerase is surprisingly nonspecific: The enzyme can add an A residue to immature tRNA-NCC in vivo (19), augmenting tRNA repair by the CCA-adding enzyme, but it can also add a poly(A) tail to mature tRNA in vitro (25) (SI Fig. 10B).…”

“…In most eubacteria, CCA addition is carried out by a single CCA-adding enzyme, but in the deeply branching eubacteria Aquifex aeolicus and Deinococcus radiodurans (17,18), and possibly in the Gram-positive eubacterium Bacillus halodurans (19), related CC-and A-adding enzymes work sequentially to add CCA. All of these eubacterial CCA-, CC-, and A-adding enzymes, as well as eubacterial poly(A) polymerase and eukaryotic CCA-adding enzymes, belong to the same class II NTR superfamily (6,17,19,20); moreover, a sequence alignment of the nucleotide binding region (helices F and G) in class II enzymes (SI Fig. 5) reveals that the protein template residues in the BstCCA enzyme (E153, D154, and R157) are conserved in the A. aeolicus CC-adding enzyme (E164, D165, and R168) and A-adding enzyme (E583, D584, and R587).…”

Characterization of tRNACys processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control

“…In organelles, the tRNA nucleotidyltransferase only has to add two nucleotides, C and A, to the 39 end because a high number of plastid tRNA genes encode a C at the 39 end (Mayer et al, 2000;Tomita et al, 1996). Aquifex aeolicus, Deinococcus radiodurans, some cyanobacteria and recently Bacillus halodurans, Bacillus clausii and Geobacter sulfurreducens have been found to contain separate enzymes for the addition of A and C residues to tRNA in vivo (Bralley et al, 2005(Bralley et al, , 2009Neuenfeldt et al, 2008;Seth et al, 2002;Tomita & Weiner, 2001. In Streptomyces coelicolor less than a third of the tRNA genes encode the CCA end and the product of SCO3896, the S. coelicolor CCase, is essential in this process (Bralley et al, 2006).…”

“…In some organisms a single CCase modifies the 39 end of damaged or immature tRNAs in a template-independent manner. Interestingly, it has been shown that in Aquifex aeolicus (Tomita & Weiner, 2001), Synechocystis sp., Deinococcus radiodurans (Tomita & Weiner, 2001), Bacillus halodurans (Bralley et al, 2005), Bacillus clausii (Neuenfeldt et al, 2008) and Geobacter sulfurreducens (Bralley et al, 2009) there are two enzymes, one of which adds the CC dinucleotide and one that adds the terminal A. In B. subtilis there is only a single gene coding for CCase (Raynal et al, 1998).…”

“…The enzymes involved in the addition of CCA, ATP(CTP) : tRNA nucleotidyltransferases (CCases), belong to a nucleotidyltransferase superfamily and are highly conserved (Bralley et al, 2005). In some organisms a single CCase modifies the 39 end of damaged or immature tRNAs in a template-independent manner.…”