Recent studies on endogenous SsrA-tagged proteins have revealed that the tagging could occur at a position corresponding to the normal termination codon. During the study of SsrA-mediated LacI tagging (Abo et al., EMBO J, 2000 19:3762–3769), we found that a variant LacI (LacIΔC1) lacking the last C-terminal amino acid residue is efficiently tagged in a stop codon-dependent manner. SsrA tagging of LacIΔC1 occurred efficiently without LacI binding to the lac operators at any one of three stop codons. The C-terminal (R)LESG peptide of LacIΔC1 was shown to trigger the SsrA tagging of an unrelated protein (CRP) when fused to its C terminus. Mass spectrometry analysis of the purified fusion proteins revealed that SsrA tagging occurs at a position corresponding to the termination codon. The alteration of the amino acid sequence but not the nucleotide sequence of the C-terminal portion eliminated the tagging. We also showed that the tagging-provoking sequences cause an efficient translational readthrough at UGA but not UAA codons. In addition, we found that C-terminal dipeptides known to induce an efficient translation readthrough could cause an efficient tagging at stop codons. We conclude that the amino acid sequence of nascent polypeptide prior to stop codons is a major determinant for the SsrA tagging at all three stop codons.

In this communication, we describe a simple and reliable method for RNA structure determination in vivo, using the divalent ion, lead(II), as a structural probe. Lead(II) is known to cleave RNA within single-stranded regions, loops, and bulges, whereas cleavages in double-stranded regions are weaker or absent. Because the ion easily entered bacterial cells, Escherichia coli cultures were treated by addition of 50–100 mM lead(II) acetate for 3–7 min, resulting in partial cleavage of RNA in vivo. Cleavage positions were mapped by reverse transcription analysis of total extracted RNA. Three RNAs were analyzed: tmRNA, CopT (the target of the antisense RNA CopA), and the leader region of the ompF mRNA. All three RNAs had previously been analyzed in vitro, and secondary structure models were available. The results presented here show that lead(II) cleavages in vivo yield detailed structural information for these RNAs, which was in good agreement with the models proposed based on in vitro work. These data illustrate the potential of lead(II) as a sequence-independent RNA structure probe for use in living cells.

tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans-translation reaction that contributes to the recycling of stalled ribosomes at the 3′ end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperature-sensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells (ΔssrA) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of ΔssrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the ΔssrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics.

DNA harvested directly from complex natural microbial communities by PCR has been successfully used to predict RNase P RNA structure, and can potentially provide an abundant source of information for structural predictions of other RNAs. In this study, we utilized genetic variation in natural communities to test and refine the secondary and tertiary structural model for the bacterial tmRNA. The variability of proposed tmRNA secondary structures in different organisms and the lack of any predicted tertiary structure suggested that further refinement of the tmRNA could be useful. To increase the phylogenetic representation of tmRNA sequences, and thereby provide additional data for statistical comparative analysis, we amplified, sequenced, and compared tmRNA sequences from natural microbial communities. Using primers designed from gamma proteobacterial sequences, we determined 44 new tmRNA sequences from a variety of environmental DNA samples. Covariation analyses of these sequences, along with sequences from cultured organisms, confirmed most of the proposed tmRNA model but also provided evidence for a new tertiary interaction. This approach of gathering sequence information from natural microbial communities seems generally applicable in RNA structural analysis.

tmRNA facilitates a novel translation, trans-translation, in which a ribosome can switch between translation of a truncated mRNA and the tmRNA's tag sequence. The mechanism underlying resumption of translation at a definite position is not known. In the present study, the effects of mutations around the initiation point of the tag-encoding sequence of Escherichia coli tmRNA on the efficiency and the frame of tag translation were assessed by measuring the incorporations of several amino acids into in vitro poly (U)-dependent tag-peptide synthesis. One-nucleotide insertions within the tag-encoding region did not shift the frame of tag translation. Any 1-nt deletion within the span of −5 to −1, but not at −6, made the frame of tag translation heterologous. Positions at which a single base substitution caused a decrease of trans-translation efficiency were concentrated within the span of −4 to −2. In particular, an A-4 to C-4 mutation seriously damaged the trans-translation, although this mutant retained normal aminoacylation and ribosome-binding abilities. A possible stem and loop structure around this region was not required for trans-translation. It was concluded that the tag translation requires the primary sequence encompassing −6 to +1, in which the central 3 nt, A-4, G-3, and U-2, play an essential role. It was also found that several base substitutions within the span of −6 to −1 extensively shifted the tag-initiation point by −1.

The conformation of the Escherichia coli 10Sa RNA (tmRNA) in solution was investigated using chemical and enzymatic probes. Single- and double-stranded domains were identified by hydrolysis of tmRNA in imidazole buffer and by lead(II)-induced cleavages. Ribonucleases T1 and S1 were used to map unpaired nucleotides and ribonuclease V1 was used to identify paired bases or stacked nucleotides. Specific atomic positions of bases were probed with dimethylsulfate, a carbodiimide, and diethylpyrocarbonate. Covariations, identified by sequence alignment with nine other tmRNA sequences, suggest the presence of several tertiary interactions, including pseudoknots. Temperature-gradient gel electrophoresis experiments showed structural transitions of tmRNA starting around 40°C, and enzymatic probing performed at selected temperatures revealed the progressive melting of several predicted interactions.

Based on these data, a secondary structure is proposed, containing two stems, four stem-loops, four pseudoknots, and an unstable structural domain, some connected by single-stranded A-rich sequence stretches. A tRNA-like domain, including an already reported acceptor branch, is supported by the probing data. A second structural domain encompasses the coding sequence, which extends from the top of one stem-loop to the top of another, with a 7-nt single-stranded stretch between. A third structural module containing pseudoknots connects and probably orients the tRNA-like domain and the coding sequence. Several discrepancies between the probing data and the phylogeny suggest that E. coli tmRNA undergoes a conformational change.