tRNA/mRNA Mimicry by tmRNA and SmpB in Trans-Translation

Since accurate translation from mRNA to protein is critical to survival, cells have developed translational quality control systems. Bacterial ribosomes stalled on truncated mRNA are rescued by a system involving tmRNA and SmpB referred to as trans-translation. Here, we review current understanding of the mechanism of trans-translation. Based on results obtained by using directed hydroxyl radical probing, we propose a new type of molecular mimicry during trans-translation. Besides such chemical approaches, biochemical and cryo-EM studies have revealed the structural and functional aspects of multiple stages of trans-translation. These intensive works provide a basis for studying the dynamics of tmRNA/SmpB in the ribosome

Probing the structure of the Escherichia coli 10Sa RNA (tmRNA)

Journal ArticleThe 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 degrees 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

Bacterial Ribosome Rescue Systems

To maintain proteostasis, the cell employs multiple ribosome rescue systems to relieve the stalled ribosome on problematic mRNA. One example of problematic mRNA is non-stop mRNA that lacks an in-frame stop codon produced by endonucleolytic cleavage or transcription error. In Escherichia coli, there are at least three ribosome rescue systems that deal with the ribosome stalled on non-stop mRNA. According to one estimation, 2–4% of translation is the target of ribosome rescue systems even under normal growth conditions. In the present review, we discuss the recent findings of ribosome rescue systems in bacteria

Role of the C-terminal tail of SmpB in the early stage of trans-translation

Trans-translation relieves a stalled translation on the bacterial ribosome by transfer-messenger RNA (tmRNA) with the help of SmpB, an essential cofactor of tmRNA. Here, we examined the role of the unstructured C-terminal tail of SmpB using an in vitro trans-translation system. It was found that truncation of the C-terminal tail or substitution of tryptophan residue at 147 in the middle of the C-terminal tail affected the activity in the early stage of trans-translation. Our investigations also revealed that the C-terminal tail is not required for the events until GTP is hydrolyzed by EF-Tu in complex with tmRNA-SmpB. A synthetic peptide corresponding to the C-terminal tail of SmpB inhibited peptidyl-transfer of alanyl-tmRNA and A-site binding of SmpB, but not GTP hydrolysis. These results suggest that the C-terminal tail has a role in the step of accommodation of alanyl-tmRNA-SmpB into the A-site. Directed hydroxyl radical probing indicated that tryptophan residue at 147 is located just downstream of the decoding center in the mRNA path when SmpB is in the A-site

Impairment of ribosome maturation or function confers salt resistance on Escherichia coli cells.

We found that loss of integrity of the ribosome by removal of a putative ribosome maturation factor or a ribosomal protein conferred salt tolerance on Escherichia coli cells. Some protein synthesis inhibitors including kasugamycin and chloramphenicol also had a similar effect, although kasugamycin affected neither 16S rRNA maturation nor subunit association into a 70S ribosome. Thus, salt tolerance is a common feature of cells in which maturation or function of the ribosome is impaired. In these cells, premature induction of an alternative sigma factor, σ(E), by salt stress was observed. These results suggest the existence of a yet-unknown stress response pathway mediated by the bacterial ribosome