The Bacillus subtilis Nucleotidyltransferase Is a tRNA CCA-Adding Enzyme

Abstract

There has been increased interest in bacterial polyadenylation with the recent demonstration that 3 poly(A) tails are involved in RNA degradation. Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) family that includes the functionally related tRNA CCA-adding enzymes. Thirty members of the Ntr family were detected in a search of the current database of eubacterial genomic sequences. Gram-negative organisms from the ␤ and ␥ subdivisions of the purple bacteria have two genes encoding putative Ntr proteins, and it was possible to predict their activities as either PAP or CCA adding by sequence comparisons with the E. coli homologues. Prediction of the functions of proteins encoded by the genes from more distantly related bacteria was not reliable. The Bacillus subtilis papS gene encodes a protein that was predicted to have PAP activity. We have overexpressed and characterized this protein, demonstrating that it is a tRNA nucleotidyltransferase. We suggest that the papS gene should be renamed cca, following the notation for its E. coli counterpart. The available evidence indicates that cca is the only gene encoding an Ntr protein, despite previous suggestions that B. subtilis has a PAP similar to E. coli PAP I. Thus, the activity involved in RNA 3 polyadenylation in the gram-positive bacteria apparently resides in an enzyme distinct from its counterpart in gram-negative bacteria.

Poly(A) polymerase activity and RNA polyadenylation in Streptomyces coelicolor A3(2)

“…The corresponding value reported for E. coli PcnB is 53 871 (Cao and Sarkar, 1992). It should be noted that ORF H24.18 shows even greater similarity to a protein from Bacillus subtilis than to E. coli PAP, but that gene, originally designated papS , has since been shown to encode a tRNA nucleotidyl transferase (Raynal et al ., 1998). A more extensive comparison of putative pap and tRNA nucleotidyl transferase genes is provided by Raynal et al .…”

Poly(A) polymerase activity and RNA polyadenylation in Streptomyces coelicolor A3(2)

“…The corresponding value reported for E. coli PcnB is 53 871 (Cao and Sarkar, 1992). It should be noted that ORF H24.18 shows even greater similarity to a protein from Bacillus subtilis than to E. coli PAP, but that gene, originally designated papS , has since been shown to encode a tRNA nucleotidyl transferase (Raynal et al ., 1998). A more extensive comparison of putative pap and tRNA nucleotidyl transferase genes is provided by Raynal et al .…”

Poly(A) polymerase I of Escherichia coli: characterization of the catalytic domain, an RNA binding site and regions for the interaction with proteins involved in mRNA degradation

“…The eubacterial nucleotidyltransferases are a family of proteins related to the E. coli PAP I (Raynal et al ., 1998). Bacteria, such as Synnechocystis, that are separated in evolutionary time from E. coli by as much as 1600 million years encode related proteins.…”

“…Sequence alignment showing three highly conserved motifs in the putative catalytic core of the eubacterial nucleotidyltransferases. EcoPAP , E. coli PAP I; EcoCCA , E. coli tRNA CCA‐adding enzyme; Ctr , C. trachomatis (encoding two enzymes designated 1 and 2 whose function cannot be predicted by sequence comparison); BsuCCA, B. subtilis tRNA CCA‐adding enzyme (also known as PapS, but see Raynal et al ., 1998); Spy , S. pyogene ; Bbu , B. burgdorferi ; Hpy , H. pyroli ; Mtu , M. tuberculosis ; Aqu , Aquifex ; and Syn , Synnechocystis . For the enzymes not designated by either PAP or CCA, their function remains to be elucidated.…”

“…We have recently demonstrated that an Ntr from B. subtilis , which was predicted to be a PAP, is actually a tRNA nucleotidyltransferase (Raynal et al ., 1998). Only in the Gram‐negative organisms from the β‐ and γ‐subdivision of the purple bacteria, which includes E. coli , is it possible to predict function by sequence comparisons.…”

Found in translation: CCA-adding enzymes reveal bacterialization of Asgard archaea prior to eukaryogenesis

“…CCA2 identification in bacteria. an experimentally validated CCA-adding enzyme from Bacillus subtilis strain 168 (UniProt accession P42977) 45 was used as the query sequence for tblastn searches against all bacterial genomes. This protein has been biochemically characterized and confirmed as a functional Class 2 CCA-adding enzyme.…”

Poly(A) polymerase activity and RNA polyadenylation in Streptomyces coelicolor A3(2)

“…The corresponding value reported for E. coli PcnB is 53 871 (Cao and Sarkar, 1992). It should be noted that ORF H24.18 shows even greater similarity to a protein from Bacillus subtilis than to E. coli PAP, but that gene, originally designated papS , has since been shown to encode a tRNA nucleotidyl transferase (Raynal et al ., 1998). A more extensive comparison of putative pap and tRNA nucleotidyl transferase genes is provided by Raynal et al .…”

Poly(A) polymerase I of Escherichia coli: characterization of the catalytic domain, an RNA binding site and regions for the interaction with proteins involved in mRNA degradation

“…The eubacterial nucleotidyltransferases are a family of proteins related to the E. coli PAP I (Raynal et al ., 1998). Bacteria, such as Synnechocystis, that are separated in evolutionary time from E. coli by as much as 1600 million years encode related proteins.…”

“…Sequence alignment showing three highly conserved motifs in the putative catalytic core of the eubacterial nucleotidyltransferases. EcoPAP , E. coli PAP I; EcoCCA , E. coli tRNA CCA‐adding enzyme; Ctr , C. trachomatis (encoding two enzymes designated 1 and 2 whose function cannot be predicted by sequence comparison); BsuCCA, B. subtilis tRNA CCA‐adding enzyme (also known as PapS, but see Raynal et al ., 1998); Spy , S. pyogene ; Bbu , B. burgdorferi ; Hpy , H. pyroli ; Mtu , M. tuberculosis ; Aqu , Aquifex ; and Syn , Synnechocystis . For the enzymes not designated by either PAP or CCA, their function remains to be elucidated.…”

“…We have recently demonstrated that an Ntr from B. subtilis , which was predicted to be a PAP, is actually a tRNA nucleotidyltransferase (Raynal et al ., 1998). Only in the Gram‐negative organisms from the β‐ and γ‐subdivision of the purple bacteria, which includes E. coli , is it possible to predict function by sequence comparisons.…”

Found in translation: CCA-adding enzymes reveal bacterialization of Asgard archaea prior to eukaryogenesis

“…CCA2 identification in bacteria. an experimentally validated CCA-adding enzyme from Bacillus subtilis strain 168 (UniProt accession P42977) 45 was used as the query sequence for tblastn searches against all bacterial genomes. This protein has been biochemically characterized and confirmed as a functional Class 2 CCA-adding enzyme.…”

Poly(A) polymerase activity and RNA polyadenylation in Streptomyces coelicolor A3(2)

“…The corresponding value reported for E. coli PcnB is 53 871 (Cao and Sarkar, 1992). It should be noted that ORF H24.18 shows even greater similarity to a protein from Bacillus subtilis than to E. coli PAP, but that gene, originally designated papS , has since been shown to encode a tRNA nucleotidyl transferase (Raynal et al ., 1998). A more extensive comparison of putative pap and tRNA nucleotidyl transferase genes is provided by Raynal et al .…”

Poly(A) polymerase I of Escherichia coli: characterization of the catalytic domain, an RNA binding site and regions for the interaction with proteins involved in mRNA degradation

“…The eubacterial nucleotidyltransferases are a family of proteins related to the E. coli PAP I (Raynal et al ., 1998). Bacteria, such as Synnechocystis, that are separated in evolutionary time from E. coli by as much as 1600 million years encode related proteins.…”

“…Sequence alignment showing three highly conserved motifs in the putative catalytic core of the eubacterial nucleotidyltransferases. EcoPAP , E. coli PAP I; EcoCCA , E. coli tRNA CCA‐adding enzyme; Ctr , C. trachomatis (encoding two enzymes designated 1 and 2 whose function cannot be predicted by sequence comparison); BsuCCA, B. subtilis tRNA CCA‐adding enzyme (also known as PapS, but see Raynal et al ., 1998); Spy , S. pyogene ; Bbu , B. burgdorferi ; Hpy , H. pyroli ; Mtu , M. tuberculosis ; Aqu , Aquifex ; and Syn , Synnechocystis . For the enzymes not designated by either PAP or CCA, their function remains to be elucidated.…”

“…We have recently demonstrated that an Ntr from B. subtilis , which was predicted to be a PAP, is actually a tRNA nucleotidyltransferase (Raynal et al ., 1998). Only in the Gram‐negative organisms from the β‐ and γ‐subdivision of the purple bacteria, which includes E. coli , is it possible to predict function by sequence comparisons.…”

Found in translation: CCA-adding enzymes reveal bacterialization of Asgard archaea prior to eukaryogenesis

“…CCA2 identification in bacteria. an experimentally validated CCA-adding enzyme from Bacillus subtilis strain 168 (UniProt accession P42977) 45 was used as the query sequence for tblastn searches against all bacterial genomes. This protein has been biochemically characterized and confirmed as a functional Class 2 CCA-adding enzyme.…”