UV irradiation of an in vitro translation mixture induced cross-linking of 4-thioU-substituted tmRNA to Escherichia coli ribosomes by forming covalent complexes with ribosomal protein S1 and 16S rRNA. In the absence of S1, tmRNA was unable to bind and label ribosomal components. Mobility assays on native gels demonstrated that protein S1 bound to tmRNA with an apparent binding constant of 1 3 10 8 M ±1 . A mutant tmRNA, lacking the tag coding region and pseudoknots pk2, pk3 and pk4, did not compete with full-length tmRNA, indicating that this region is required for S1 binding. This was con®rmed by identi®cation of eight cross-linked nucleotides: U85, located before the resume codon of tmRNA; U105, in the mRNA portion of tmRNA; U172 in pK2; U198, U212, U230 and U240 in pk3; and U246, in the junction between pk3 and pk4. We concluded that ribosomal protein S1, in concert with the previously identi®ed elongation factor EF-Tu and protein SmpB, plays an important role in tmRNA-mediated trans-translation by facilitating the binding of tmRNA to ribosomes and forming complexes with free tmRNA.

Minimal secondary structures of the bacterial and plastid tmRNAs were derived by comparative analyses of 50 aligned tmRNA sequences. The structures include 12 helices and four pseudoknots and are refinements of earlier versions, but include only those base pairs for which there is comparative evidence. Described are the conserved and variable features of the tmRNAs from a wide phylogenetic spectrum, the structural properties specific to the bacterial subgroups and preliminary 3-dimensional models from the pseudoknotted regions.

Protein L27 has been implicated as a constituent of the peptidyl transferase center of the Escherichia coli 50 S ribosomal subunit by a variety of experimental observations. To define better the functional role of this protein, we constructed a strain in which the rpmA gene, which encodes L27, was replaced by a kanamycin resistance marker. The deletion mutant grows five to six times slower than the wild-type parent and is both coldand temperature-sensitive. This phenotype is reversed when L27 is expressed from a plasmid-borne copy of the rpmA gene. Analysis of ribosomes from the L27-lacking strain revealed deficiencies in both the assembly and activity of the 50 S ribosomal subunits. Although functional 50 S subunits are formed in the mutant, an assembly "bottleneck" was evidenced by the accumulation of a prominent 40 S precursor to the 50 S subunit which was deficient in proteins L16, L20, and L21, as well as L27. In addition, the peptidyl transferase activity of 70 S ribosomes containing mutant 50 S subunits was determined to be three to four times lower than for wild-type ribosomes. Ribosomes lacking L27 were found to be impaired in the enzymatic binding of Phe-tRNA Phe to the A site, although the interaction of N-acetyl-Phe-tRNA Phe with the P site was largely unperturbed. We therefore infer that L27 contributes to peptide bond formation by facilitating the proper placement of the acceptor end of the A-site tRNA at the peptidyl transferase center.Protein L27 is one of the smallest and the most basic polypeptides in the Escherichia coli ribosome (1). A combination of immune electron microscopy and protein-protein crosslinking results places L27 at the base of the central protuberance on the interface side of the 50 S subunit (2). According to in vitro studies, L27 is a late assembly protein (3) and does not have an identifiable binding site on the 23 S rRNA. Chemical and UV cross-linking studies, however, demonstrated that L27 is associated closely with domain V of the 23 S rRNA (4 -6), a region that comprises part of the peptidyl transferase center (for review, see Ref. 7). The proximity of L27 to the peptidyl transferase center was also supported by affinity labeling studies with inhibitors of peptidyl transferase activity, such as chloramphenicol, carbomycin, tylosin, and spiramycin (8 -11), as well as with puromycin (12), an antibiotic that mimics the aminoacyl-adenosine moiety of aminoacyl-tRNA and is a substrate for peptidyl transferase (13). Direct evidence for the presence of L27 at the peptidyl transferase center was obtained through the use of derivatives of tRNA Phe containing photoreactive azidonucleotides within the 3Ј-terminal ACCA OH sequence (14,15). When bound to the ribosomal A or P site and irradiated, these probes became cross-linked predominantly to protein L27 and domain V of 23 S rRNA (15, 16).The contribution of protein L27 to the peptidyl transferase center has been addressed by a number of studies. It has been found, for instance, that the omission of L27 during in vitro reconstituti...

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...

When E. coli ribosomal subunits are reacted with 2-iminothiolane and then subjected to a mild ultraviolet irradiation, an RNA-protein cross-linking reaction occurs. About 5% of the total protein in each subunit becomes cross-linked to the RNA, and a specific sub-set of proteins is involved in the reaction. In the case of the 50S subunit, the sites of cross-linking to the 23S RNA have been determined for six of these proteins: protein L4 is cross-linked within an oligonucleotide comprising positions 613-617 in the 23S sequence, L6 within positions 2473-2481, L21 within positions 540-548, L23 within positions 137-141, L27 within positions 2332-2337 and L29 within positions 99-107.