Contributions of Pseudoknots and Protein SmpB to the Structure and Function of tmRNA in trans-Translation

Abstract

Bacteria contain transfer-messenger RNA (tmRNA), a molecule that during trans-translation tags incompletely translated proteins with a small peptide to signal the proteolytic destruction of defective polypeptides. TmRNA is composed of tRNA-and mRNA-like domains connected by several pseudoknots. Using truncated ribosomal protein L27 as a reporter for tagging in vitro and in vivo, we have developed exceptionally sensitive assays to study the role of Escherichia coli tmRNA in trans-translation. Site-directed mutagenesis experiments showed that pseudoknot 2 and the abutting helix 5 were particularly important for the binding of ribosomal protein S1 to tmRNA. Pseudoknot 4 not only facilitated tmRNA maturation but also promoted tagging. In addition, the three pseudoknots (pk2 to pk4) were shown to play a significant role in the proper folding of the tRNA-like domain. Protein SmpB enhanced tmRNA processing, suggesting a new role for SmpB in transtranslation. Taken together, these results provide unanticipated insights into the functions of the pseudoknots and protein SmpB during tmRNA folding, maturation, and protein synthesis.An interruption of the elongation step of protein synthesis results in the production of truncated proteins and leaves ribosomes stalled at the 3Ј end of mRNA templates lacking a stop codon(s). To remove the defective polypeptides and recycle the ribosomes, bacteria have developed trans-translation, a quality control mechanism that tags the C termini of defective proteins with a short peptide recognized by housekeeping proteases. This peptide tag is encoded by a short open reading frame present in a small stable RNA molecule called 10Sa or transfermessenger RNA (tmRNA).1 It has been shown that in addition to protein tagging, tmRNA facilitates the recycling of ribosomes by providing missing stop codons (1, 2).Structure probing of the Escherichia coli tmRNA and sequence comparisons demonstrated the presence of three domains (Fig. 1A) (3-5). The 3Ј and 5Ј termini of the tmRNA form the tRNA-like domain (TLD) with a significantly reduced D arm. The resume codon and stop codon(s) demarcate the open reading frame in the mRNA-like domain (MLD). The TLD and the MLD are connected by a pseudoknot-rich domain consisting of four pseudoknots (pk1 to pk4) in most tmRNAs.The three-dimensional model of E. coli tmRNA suggests a structure in which the TLD is connected to the circularly arranged MLD and pseudoknots through coaxially stacked helices (6). Recently, the entry of tmRNA into a stalled E. coli ribosome has been visualized by cryo-electron microscopy (7). At this particular step of trans-translation, the TLD, pk1, and the MLD contact the ribosome, whereas the pk2 to pk4 segment forms an arc that remains outside the ribosome.Three proteins facilitate binding of tmRNA to the ribosome. Elongation factor Tu forms a ternary complex with GTP and aminoacyl-tmRNA, as in regular protein synthesis (8, 9). Protein SmpB binds to the ternary complex in vitro as well as to stalled ribosomes in vivo (10 -12). Ribosomal p...

Ribosomal Protein S1: An Important Trans-Translational Factor

“…MS2-tmRNA(H8), a variant of E. coli tmRNAH8 that can bind the coat protein from MS2 bacteriophage, was constructed by PCRdirected mutagenesis, as described earlier [17]. Two mutants, called MS2-tmRNA-4 and MS2-tmRNA-7 were prepared ( Figure 1B).…”

“…coli strain IW363 was transformed with plasmid pETrpmA-At-3, which carries one copy of an rpmA gene encoding a truncated ribosomal protein L27, one copy of SmpB gene and one copy of modified ssrA gene, encoding either MS2-tmRNA-4 or -7 [17]. Synthesis of truncated ribosomal protein L27 and following tagging activity was induced by addition of 1 mM IPTG to logarithmically growing cells.…”

“…The liquid culture was incubated at 37°C for 3 hr. The expression of truncated protein L27 and its tagging were analyzed by SDS-Polyacrylamide Electrophoresis (SDS-PAGE) [17].…”

“…The tmRNA:ribosome complexes were resuspended in 1 mL of binding buffer and stored at -80°C. Protein composition of the purified tmRNA:ribosome complexes was analyzed by SDS-PAGE, as described earlier [17].…”

tmRNA on its way through the ribosome

“…SmpB is indispensable for all activities of tmRNA. [4][5][6][7][8][9][10][11] The ORF constitutes an essential segment of the mRNA-like domain (MLD) and encodes 10 residues of a degradation tag in E. coli. The latter connects to the TLD through four pseudoknots, designated PK1 through PK4.…”