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 beta and gamma 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.

FIG. 1

(A) Repair of tRNA CCA ends by nucleotidyltransferase; (B) 3′ poly(A) addition to tRNA or other RNA substrates by PAP; (C) phylogenetic tree showing the relationship of the major families of eubacteria (38). Thermus thermophilus and Aquifex are single organisms.

FIG. 2

Sequence alignment of the tRNA nucleotidyltransferase (CCA) and PAP I (PAP) homologues from members of the β and γ subdivisions of the purple bacteria (E. col, E. coli; A. hyd, A. hydrophila; A. act, Actinobacillus actinomycetemcomitans; H. inf, H. influenzae; P. aer, Pseudomonas aeruginosa; N. gon, Neisseria gonorrhoeae; N. nem, Neisseria meningitidis) with the related protein from B. subtilis (B. sub). The initial alignment was made with CLUSTAL (24, 25), and small realignments were made manually. The colored boxes indicate the positions of conserved amino acids of the CCA-adding family (blue), the PAP I family (pink), and both families (yellow). The asterisks show the conserved G-D-D-D residues that are the signature of the X polymerase family. The protein motifs used to design the degenerate inosine-containing oligonucleotides are underlined. The numbers in parentheses represent amino acids not shown in the alignment, and dashes represent gaps in the alignment.

FIG. 3

Expression and Western blot analysis of the B. subtilis PapS protein. Proteins were separated by SDS-polyacrylamide gel electrophoresis, and the gel was stained with Coomassie blue (A) or analyzed by Western blotting with the E. coli PAP I antibody (B). Lanes 1 and 2, E. coli and B. subtilis total proteins. Lanes 3 to 5, extracts of PAP I, E. coli tRNA nucleotidyltransferase, and B. subtilis PapS, respectively, were loaded to give similar amounts of each of the overexpressed proteins (see Materials and Methods). The dots in lanes 3 to 5 indicate the positions of the overexpressed proteins. The arrows in lanes 2 show the positions of a B. subtilis protein that cross-reacts with the PAP I antibody.

FIG. 4

Polyacrylamide gel analysis of products under CCA assay conditions with yeast tRNA as the acceptor and [α-³²P]ATP (A) or [α-³²P]CTP (B) as the substrate. Lanes: 1, E. coli PAP I; 2, protein extract from BL21(DE3) with the pET11a expression vector; 3, E. coli tRNA nucleotidyltransferase; 4, B. subtilis PapS.

FIG. 5

PCR amplification of the papS gene from B. subtilis genomic DNA with degenerate inosine-containing oligonucleotides designed to bind to DNA encoding two of the most highly conserved motifs in the eubacterial Ntr protein family (Fig. 2). Lane 1, B. subtilis genomic DNA; lane 2, cloned E. coli pcnB gene (in pET11a).