Closely Related CC- and A-adding Enzymes Collaborate to Construct and Repair the 3′-Terminal CCA of tRNA in Synechocystis sp. and Deinococcus radiodurans

Abstract

The 3-terminal CCA sequence of tRNA is faithfully constructed and repaired by the CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) using CTP and ATP as substrates but no nucleic acid template. Until recently, all CCA-adding enzymes from all three kingdoms appeared to be composed of a single kind of polypeptide with dual specificity for adding both CTP and ATP; however, we recently found that in Aquifex aeolicus, which lies near the deepest root of the eubacterial 16 S rRNA-based phylogenetic tree, CCA addition represents a collaboration between closely related CCadding and A-adding enzymes (Tomita, K. and Weiner, A. M. (2001) Science 294, 1334 -1336). Here we show that in Synechocystis sp. and Deinococcus radiodurans, as in A. aeolicus, CCA is added by homologous CC-and A-adding enzymes. We also find that the eubacterial CCA-, CC-, and A-adding enzymes, as well as the related eubacterial poly(A) polymerases, each fall into phylogenetically distinct groups derived from a common ancestor. Intriguingly, the Thermatoga maritima CCA-adding enzyme groups with the A-adding enzymes, suggesting that these distinct tRNA nucleotidyltransferase activities can intraconvert over evolutionary time.The 3Ј-terminal CCA sequence (positions 74 -76 in the standard cloverleaf representation) is universally present in the tRNAs of all organisms (1) and is important for many aspects of gene expression. The CCA terminus is required for the aminoacylation of tRNA (2, 3) and for translation on the ribosome where the CCA sequences of the aminoacyl-and peptidyl-tRNA pair with the large ribosomal RNA near the peptidyltransferase center (4 -6). In eubacteria, the CCA sequence is required for the efficient maturation of the 5Ј-end of tRNA by RNase P (7,8). In eukaryotes, the CCA sequence serves as an antideterminant to block 3Ј-exonuclease activity (9), and it is essential for the export of mature tRNA from the nucleus to the cytoplasm (10, 11).The CCA-adding enzyme (ATP(CTP):tRNA nucleotidyltransferase) builds and repairs the 3Ј-terminal CCA sequence of all tRNAs (12). CCA-adding activity has been identified in all three kingdoms, suggesting conservation of function and perhaps structure throughout evolution (13). CCA-adding activity is essential in some eubacteria as well as in all archaea and eukaryotes where some or all tRNA genes do not encode CCA (14). Yet, even in organisms such as Escherichia coli where all tRNA genes do encode CCA, CCA-adding activity confers a substantial selective advantage, probably by repairing tRNAs that have been subject to errant nucleolytic attack (15).The CCA-adding enzyme belongs to the nucleotidyltransferase (NTR) 1 family, a large protein superfamily that encompasses template-dependent DNA polymerases (DNA polymerase ␤) and template-independent RNA and DNA polymerases (poly(A) polymerase, terminal deoxynucleotidyltransferase, and CCA-adding enzymes) as well as metabolic regulators (GlnB uridylyltransferase, glutamine synthase adenylyltransferase) and antibiotic resistance factors (kanamycin nucleo...

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

“…We speculate that the low levels of activity observed for NTSFI with ATP as a substrate (lane 4) and for NTSFII with CTP as a substrate (lane 9) reflect relaxed specificities of the two enzymes in vitro. Thus, our results are consistent with the conclusion that, like A. aeolicus, D. radiodurans, and at least some cyanobacteria (32,34,35), B. halodurans contains separate enzymes for the addition of A and C residues to tRNA in vivo. It is possible that the smear above the major band of lane 8 may reflect the synthesis of poly(C), as has been observed for other TNTs (14,32).…”

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

“…We speculate that the low levels of activity observed for NTSFI with ATP as a substrate (lane 4) and for NTSFII with CTP as a substrate (lane 9) reflect relaxed specificities of the two enzymes in vitro. Thus, our results are consistent with the conclusion that, like A. aeolicus, D. radiodurans, and at least some cyanobacteria (32,34,35), B. halodurans contains separate enzymes for the addition of A and C residues to tRNA in vivo. It is possible that the smear above the major band of lane 8 may reflect the synthesis of poly(C), as has been observed for other TNTs (14,32).…”

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

“…We speculate that the CCA-adding TNT activity observed in the beta-and gammaproteobacteria evolved via mutations in the genes encoding either the C-or A-adding TNTs of an ancestral species and that subsequent mutations resulted in the conversion of the CCA-adding TNT to a PAP. The apparent absence of a PAP I-like activity in the so-called deeply branching bacterial species, such as Aquifex, Thermotoga, and Deinococcus species, and the cyanobacteria (1,22,23) is consistent with the hypothesis that polyadenylation of RNA 3Ј tails by a PAP I-like enzyme evolved subsequent to the evolution of TNTs. The G. sulfurreducens PAP gene and the PAP I genes of the beta-and gammaproteobacteria may have arisen via independent evolutionary events.…”

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

“…Similarly unexpected is the observation that k cat /K m for GTP addition by the G-adding enzyme is actually 5.3-fold improved relative to k cat /K m for ATP addition by the wild-type A-adding enzyme (SI Table 2). Taken together, these observations are more consistent with duplication of a CCA-adding gene to give CC-and A-adding activities than with evolution of a CCor A-adding enzyme into a true CCA-adding activity as originally proposed (18). The decreased catalytic efficiency of the A. aeolicus CC-and A-adding enzymes might then reflect the compromises required for the new nucleotide binding sites to exclude one NTP when the enzyme originally bound both, and for the new tRNA binding sites to exclude tRNAs of nearly comparable length when the site originally bound three different substrates (tRNA-N, tRNA-NC, and tRNA-NCC).…”

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

“…We speculate that the low levels of activity observed for NTSFI with ATP as a substrate (lane 4) and for NTSFII with CTP as a substrate (lane 9) reflect relaxed specificities of the two enzymes in vitro. Thus, our results are consistent with the conclusion that, like A. aeolicus, D. radiodurans, and at least some cyanobacteria (32,34,35), B. halodurans contains separate enzymes for the addition of A and C residues to tRNA in vivo. It is possible that the smear above the major band of lane 8 may reflect the synthesis of poly(C), as has been observed for other TNTs (14,32).…”

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

“…We speculate that the CCA-adding TNT activity observed in the beta-and gammaproteobacteria evolved via mutations in the genes encoding either the C-or A-adding TNTs of an ancestral species and that subsequent mutations resulted in the conversion of the CCA-adding TNT to a PAP. The apparent absence of a PAP I-like activity in the so-called deeply branching bacterial species, such as Aquifex, Thermotoga, and Deinococcus species, and the cyanobacteria (1,22,23) is consistent with the hypothesis that polyadenylation of RNA 3Ј tails by a PAP I-like enzyme evolved subsequent to the evolution of TNTs. The G. sulfurreducens PAP gene and the PAP I genes of the beta-and gammaproteobacteria may have arisen via independent evolutionary events.…”

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

“…Similarly unexpected is the observation that k cat /K m for GTP addition by the G-adding enzyme is actually 5.3-fold improved relative to k cat /K m for ATP addition by the wild-type A-adding enzyme (SI Table 2). Taken together, these observations are more consistent with duplication of a CCA-adding gene to give CC-and A-adding activities than with evolution of a CCor A-adding enzyme into a true CCA-adding activity as originally proposed (18). The decreased catalytic efficiency of the A. aeolicus CC-and A-adding enzymes might then reflect the compromises required for the new nucleotide binding sites to exclude one NTP when the enzyme originally bound both, and for the new tRNA binding sites to exclude tRNAs of nearly comparable length when the site originally bound three different substrates (tRNA-N, tRNA-NC, and tRNA-NCC).…”

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

“…We speculate that the low levels of activity observed for NTSFI with ATP as a substrate (lane 4) and for NTSFII with CTP as a substrate (lane 9) reflect relaxed specificities of the two enzymes in vitro. Thus, our results are consistent with the conclusion that, like A. aeolicus, D. radiodurans, and at least some cyanobacteria (32,34,35), B. halodurans contains separate enzymes for the addition of A and C residues to tRNA in vivo. It is possible that the smear above the major band of lane 8 may reflect the synthesis of poly(C), as has been observed for other TNTs (14,32).…”

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

“…We speculate that the CCA-adding TNT activity observed in the beta-and gammaproteobacteria evolved via mutations in the genes encoding either the C-or A-adding TNTs of an ancestral species and that subsequent mutations resulted in the conversion of the CCA-adding TNT to a PAP. The apparent absence of a PAP I-like activity in the so-called deeply branching bacterial species, such as Aquifex, Thermotoga, and Deinococcus species, and the cyanobacteria (1,22,23) is consistent with the hypothesis that polyadenylation of RNA 3Ј tails by a PAP I-like enzyme evolved subsequent to the evolution of TNTs. The G. sulfurreducens PAP gene and the PAP I genes of the beta-and gammaproteobacteria may have arisen via independent evolutionary events.…”

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

“…Similarly unexpected is the observation that k cat /K m for GTP addition by the G-adding enzyme is actually 5.3-fold improved relative to k cat /K m for ATP addition by the wild-type A-adding enzyme (SI Table 2). Taken together, these observations are more consistent with duplication of a CCA-adding gene to give CC-and A-adding activities than with evolution of a CCor A-adding enzyme into a true CCA-adding activity as originally proposed (18). The decreased catalytic efficiency of the A. aeolicus CC-and A-adding enzymes might then reflect the compromises required for the new nucleotide binding sites to exclude one NTP when the enzyme originally bound both, and for the new tRNA binding sites to exclude tRNAs of nearly comparable length when the site originally bound three different substrates (tRNA-N, tRNA-NC, and tRNA-NCC).…”