Geobacter sulfurreducens Contains Separate C- and A-Adding tRNA Nucleotidyltransferases and a Poly(A) Polymerase

Abstract

The genome of Geobacter sulfurreducens contains three genes whose sequences are quite similar to sequences encoding known members of an RNA nucleotidyltransferase superfamily that includes tRNA nucleotidyltransferases and poly(A) polymerases. Reverse transcription-PCR using G. sulfurreducens total RNA demonstrated that the genes encoding these three proteins are transcribed. These genes, encoding proteins designated NTSFI, NTSFII, and NTSFIII, were cloned and overexpressed in Escherichia coli. The corresponding enzymes were purified and assayed biochemically, resulting in identification of NTSFI as a poly(A) polymerase, NTSFII as a C-adding tRNA nucleotidyltransferase, and NTSFIII as an A-adding tRNA nucleotidyltransferase. Analysis of G. sulfurreducens rRNAs and mRNAs revealed the presence of heteropolymeric RNA 3 tails. This is the first characterization of a bacterial system that expresses separate C-and A-adding tRNA nucleotidyltransferases and a poly(A) polymerase.RNA nucleotidyltransferases play an important role in the maturation, processing, and degradation of RNAs. tRNA nucleotidyltransferases (TNTs) and poly(A) polymerases (PAPs) are both members of a superfamily of nucleotidyltransferases (13, 27). TNTs add C and A residues to the 3Ј ends of immature or damaged tRNA molecules and are found in bacteria, archaea, and eukaryotes (24). PAPs, which are present in both eukaryotes and bacteria, polyadenylate RNA 3Ј ends (27). With regard to the distribution of RNA nucleotidyltransferases in bacteria, with only a few exceptions, the genomes of the alpha-and epsilonproteobacteria and of the low-and high-GϩC-content gram-positive bacteria appear to encode only one member of this superfamily, presumably a CCA-adding TNT (1). Several of the so-called deeply branching bacterial species, including Aquifex aeolicus and Deinococcus radiodurans, and some cyanobacteria contain two RNA nucleotidyltransferases, which have been shown to function as separate C-and A-adding TNTs (22,23). Interestingly, at least one low-GϩC-content gram-positive species, Bacillus halodurans, has also been shown to contain separate C-and A-adding TNTs (1). The beta-and gammaproteobacteria, such as Escherichia coli, also contain two RNA nucleotidyltransferases, a CCA-adding TNT and a PAP, PAP I, that adds 3Ј tails to the ends of RNA molecules in cells of these organisms (8,11,18,19). mRNAs and 16S and 23S rRNAs are polyadenylated in E. coli and presumably in other beta-and gammaproteobacteria, and polyadenylation facilitates the exonucleolytic degradation of these RNAs by RNase II and polynucleotide phosphorylase (9,16,18,19).The genomes of several deltaproteobacteria also encode two members of the RNA nucleotidyltransferase superfamily (NTSF), and the genomes of a few deltaproteobacteria appear to encode three NTSF enzymes (1). In particular, the genomes of Geobacter sulfurreducens, Geobacter metallireducens, Pelobacter carbinolicus, and Desulfotalea psychrophila all contain three open reading frames with significant homology to open reading ...

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).…”

An inhibitory C-terminal region dictates the specificity of A-adding enzymes

“…Whereas such CCA-adding enzymes are found in most organisms, several bacteria like Aquifex aeolicus, Synechocystis species, Deinococcus radiodurans, Bacillus halodurans, Geobacter sulfurreducens, and Thermus thermophilus carry two closely related class II enzymes with partial activities (11)(12)(13)(14)(15). Here, one enzyme adds two C residues to the tRNA (CC-adding enzyme), and the second nucleotidyltransferase incorporates the terminal A (A-adding enzyme).…”

tRNA nucleotidyltransferases: ancient catalysts with an unusual mechanism of polymerization

“…Interestingly, these enzymes do not synthesize the complete CCA-end, but show a restricted activity. One enzyme adds exclusively two C residues to the tRNA primer, while the second one incorporates the terminal A. Consequently, these enzymes are called CC-and A-adding enzymes, respectively [52,[72][73][74][75]. The collaboration of both activities ensures a complete and efficient CCA synthesis.…”