Significant bias against the ACA triplet in the tmRNA sequence of Escherichia coli K-12

Abstract

The toxin MazF in Escherichia coli cleaves single-stranded RNAs specifically at ACA sequences. MazF overexpression virtually eliminates all cellular mRNAs to completely block protein synthesis. However, protein synthesis can continue on an mRNA that is devoid of ACA triplets. The finding that ribosomal RNAs remain intact in the face of complete translation arrest suggested a purpose for such preservation. We therefore examined the sequences of all transcribed RNAs to determine if there was any statistically significant bias against ACA. While ACA motifs are absent from tmRNA, 4.5S RNA, and seven of the eight 5S rRNAs, statistical analysis revealed that only for tmRNA was the absence nonrandom. The introduction of single-strand ACAs makes tmRNA highly susceptible to MazF cleavage. Furthermore, analysis of tmRNA sequences from 442 bacteria showed that the discrimination against ACA in tmRNAs was seen mostly in enterobacteria. We propose that the unusual bias against ACA in tmRNA may have coevolved with the acquisition of MazF.

FIG. 1.

Location of all ACA triplets in tRNA and 5S rRNA sequences of E. coli K-12. (A) Nucleotide sequences (Seq) of any tRNAs containing ACAs are shown (for a total of 10 unique ACA-containing tRNAs), with the predicted single-strand and double-strand (Str) regions indicated by dots and arrowheads, respectively. ACA triplets are underlined, and the methylation site of is marked with an arrow. (B) The nucleotide sequence of the 5S rRNA gene, rrfF, is similarly shown.

FIG. 2.

Distribution of ACA frequencies in tmRNAs of 421 species of bacteria (A) and in tmRNAs of 39 species of enterobacteria (B) (http://www.indiana.edu/∼tmrna/). Citbr_roden, Citrobacter rodentium; Entbr_sakaz, Enterobacter sakazakii; Erwin_carot, Erwinia carotovora; Erwin_chrys, Erwinia chrysanthemi; Klebs_pneum, Klebsiellia pneumonaie; Panto_stewa, Pantoea stewartii; Phorh_lumin, Photorhabdus luminescens; Salmo_bongo, Salmonella bongori; Salmo_ente, Salmonella enterica; Serra_marc, Serratia marcescens; Shige_boydi, Shigella boydii; Shige_dyse, Shigella dysenteriae; Shige_flexn, Shigella flexneri; Shige_sonne, Shigella sonnei; Yersi_frede, Yersinia frederiksenii; Yersi_pesti, Yersinia pestis; Yersi_pseud, Yersinia pseudotuberculosis; Bloch_penns, Candidatus Blochmannia pennsylvanicus; Erwin_amylo, Erwinia amylovora; Phorh_asymb, Photorhabdus asymbiotica; Prots_mirab, Proteus mirabilis; Yersi_berco, Yersinia bercovieri; Yersi_enter, Yersinia enterocolitica; Yersi_inmed, Yersinia intermedia; Yersi_molla, Yersenia mollaretii; Bloch_flori, “Candidatus Blochmannia floridanus”; Buchn_aphi, Buchnera aphidicola; Wiggl_gloss, Wigglesworthia glossinidia.

FIG. 3.

Distribution of probabilities for the random occurrence of single-strand and double-strand ACAs in tmRNAs from 442 bacteria. The probability was calculated as described in Materials and Methods. Enterobacteria were plotted separately from other bacteria.

FIG. 4.

(A) Frequency of ACA sequences in 49 noncoding, small RNAs in E. coli K-12 (not including rRNAs and tRNAs). Numbers in parentheses are the lengths of each RNA molecule in nucleotides (nt). (B) Probability of ACA sequences in noncoding RNAs plotted by sequence length. The probability was calculated as described in Materials and Methods.

FIG. 5.

Mutant tmRNAs have tagging function. (A) Diagram of the three ACA sites introduced into E. coli K-12 tmRNA (adapted from reference with permission of the publisher). Two ACA mutations were introduced into single-strand regions, while the third was introduced into a double-strand region. (B) Radiolabeled lambda repressor is tagged by genomic tmRNA (G), wild-type (wt) tmRNA plasmid (P), each of the single tmRNA mutant plasmids (lanes 1, 2, and 3), and the tmRNA triple mutant plasmid (lane 4). The lambda repressor is not tagged (and hence smaller) in cells lacking tmRNA (−).

FIG. 6.

Induction of MazF cleaves tmRNA at single-strand ACA sites. Primer extensions were run on total RNA from strains containing pBR322 alone (−), wild-type tmRNA (wt), or triple mutant tmRNA (+aca) over a time course of 30 min. (Left) Cleavage at ACA81 (boxed) in the triple mutant but not in the wild type or vector alone. (Right) Cleavage at ACA188 in the triple mutant only but no cleavage at ACA278 with any of the vectors. Full-length (FL) primer extension products are marked with triangles. Single asterisks indicate primer extension products ending at single-strand ACA sites. The double asterisk indicates incomplete primer extension, ending at regions of stable secondary structure.

FIG. 7.

MazF cleavage occurs at ACA sites. Sequencing reactions for tmRNA were run next to primer extension reactions using total RNA harvested 0 and 30 min after MazF induction. (Left) Cleavage at A82C. (Right) Cleavage at C188A. Part of the proteolysis tag-encoding region of tmRNA is shown in brackets. Single asterisks indicate minor cleavage bands, while double asterisks indicate major cleavage bands.