Escherichia coli tmRNA lacking pseudoknot 1 tags truncated proteins in vivo and in vitro

Abstract

Transfer-messenger RNA (tmRNA) and protein SmpB facilitate trans-translation, a quality-control process that tags truncated proteins with short peptides recognized by a number of proteases and recycles ribosomes stalled at the 39 end of mRNA templates lacking stop codons. The tmRNA molecule is a hybrid of tRNA-and mRNA-like domains that are usually connected by four pseudoknots (pk1-pk4). Replacement of pk1 with a single-stranded RNA yields pk1L, a mutant tmRNA that tags truncated proteins very poorly in vitro but very efficiently in vivo. However, deletion of the whole pk1 is deleterious for protein tagging. In contrast, deletion of helix 4 yields Dh4, a fully functional tmRNA derivative containing a single hairpin instead of pk1. Further deletions in the pk1 segment yield two subclasses of mutant tmRNAs that are unable to tag truncated proteins, but some of them bind to stalled ribosomes. Our studies demonstrate that pk1 is not essential for tmRNA functions but contributes to the stability of the tmRNA structure. Our studies also indicate that the length of this RNA segment is critical for both tmRNA binding to the ribosome and resumption of translation.

Structural aspects of trans‐translation

“…Pseudoknot 1 (pK1) is positioned only 11 nucleotides upstream of the resume codon and can be involved in resume codon selection. An in vitro study supports the conclusion that pK1 is important for trans-translation (39); however, in contrast, in vivo genetic selection experiments revealed that the pK1 can be substituted with the hairpin without loss of activity (40,41). Thus, it is very likely that pK1 plays a structural role in arranging the correct positioning of the functional elements of the tmRNA.…”

Ribosomal Protein S1: An Important Trans-Translational Factor

“…Recently, we demonstrated that tmRNA lacking pseudoknot pk1 tags efficiently truncated proteins both in vitro and in vivo [37]. This observation suggests that pk1 is not a part of the protein S1 binding site on E. coli tmRNA.…”

“…We used gel mobility shift assay to see whether protein S1 binds to the pk1 transcript. As control, we used a transcript that corresponded to mutant pk1, which does not fold into a pseudoknot, but is present in the tagging-competent tmRNA Δh4/Δ1p [37]. Figure 6 shows that protein S1 binds readily to the mostly singlestranded mutant pk1, but it is not able to bind to the wild-type pk1.…”

Bacterialtrans-Translation: From Functions to Applications

“…Nonetheless, it was shown recently that the substitution of PK1 with a small and stable RNA hairpin still allows for tmRNA tagging in vitro or in vivo [44]. These data argue in favor of the participation of PK1 in stabilizing the region between the TLD and the MLD and preventing global misfolding of tmRNA, rather than having a direct role in ribosome binding [44,45]. For the three other PKs the predominant role is the folding and maturation of tmRNA [46].…”