Binding and cross-linking of tmRNA to ribosomal protein S1, on and off the Escherichia coli ribosome

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 x 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 confirmed by identification 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 identified 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.     

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

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 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.     

SmpB: a protein that binds to double-stranded segments in tmRNA and tRNA

Binding of the SmpB protein to tmRNA is essential for trans-translation, a process that facilitates peptide tagging of incompletely synthesized proteins. We have used three experimental approaches to study these interactions in vitro. Gel mobility shift assays demonstrated that tmRNA(Delta90-299), a truncated tmRNA derivative lacking pseudoknots 2-4, has the same affinity for the Escherichia coli and Aquifex aeolicus SmpB proteins as the intact E. coli tmRNA. These interactions can be challenged by double-stranded RNAs such as tRNAs and 5S rRNA and are abolished by removal of 24 amino acids from the C-terminus of the A. aeolicus protein. A combination of enzymatic probing and UV-induced cross-linking showed that three SmpB molecules can bind to a single tmRNA(Delta90-299) and tRNA molecule. Irradiation of E. coli tmRNA and yeast tRNA(Phe) bound to a single SmpB molecule with UV light induced cross-links to residues C343 and m(1)A48, respectively, in their T-loops and to their 3' terminal adenosines. These findings indicate that the acceptor-T arm constitutes the primary SmpB binding site in both tmRNA and tRNA. The remaining two SmpB molecules associate with the anticodon stem-like region of tmRNA and the anticodon arm of tRNAs. As the T and anticodon loops are dispensable for SmpB binding, it seems that SmpB recognizes double helical segments in both tmRNA and tRNA molecules. Although these interactions involve analogous elements in both molecules, their different effects on aminoacylation appear to reflect subtle structural differences between the tRNA-like domain of tmRNA and tRNA.     

Transfer-messenger RNA unfolds as it transits the ribosome

In bacteria, translation of mRNAs lacking stop codons produces truncated polypeptides and traps ribosomes in unproductive complexes. Potentially harmful truncated proteins are tagged with short peptides encoded by the mRNA-like domain of tmRNA and targeted for digestion by housekeeping proteases. We show that altered Escherichia coli transfer-messenger RNAs (tmRNAs) produce in vivo fusion proteins with peptide tags that extend far beyond the conventional termination signal of the wild-type tmRNA. Regions of tmRNA capable of serving as templates for protein synthesis include helix 5, as well as pseudoknots 2, 3, and 4. The removal of all six in-frame stop codons negatively affects tmRNA processing, thereby preventing translation of the 3' portion of the tRNA-like domain. These findings provide evidence that trans-translation can be accompanied by the unfolding of significant portions of the tmRNA molecule. Many of these conformational changes are likely to be required during trans-translation to maintain the ribosomal subunits in close proximity to the tmRNA for monitoring its transit.     

Importance of the conserved nucleotides around the tRNA-like structure of Escherichia coli transfer-messenger RNA for protein tagging

A bacterial RNA functioning as both tRNA and mRNA, transfer-messenger RNA (tmRNA) rescues stalled ribosomes and clears the cell of incomplete polypeptides. For function, Escherichia coli tmRNA requires an elaborate interplay between a tRNA-like structure and an internal mRNA domain that are connected by a 295 nt long compact secondary structure. The tRNA-like structure is surrounded by 16 unpaired nt, including 10 residues that are >95% conserved among the known 140 tmRNA sequences. All these residues were mutated to define their putative role(s) in trans-translation. Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all variants. As anticipated from the low sequence conservation, mutating positions 8–12 and position 15 affects neither aminoacylation nor protein tagging. Mutating a set of two conserved positions 13 and 14 abolishes both functions. Probing the solution conformation indicates that this defective mutant adopts an alternate conformation of its acceptor stem that is no more aminoacylatable, and thus inactive in protein tagging. Selected point mutations at the conserved nucleotide stretches 16–20 and 333–335 seriously impair protein tagging with only minor changes in their solution conformations and aminoacylation. Point mutations at conserved positions 19 and 334 abolish trans-translation and 70S ribosome binding, although retaining nearly normal aminoacylation capacities. Two proteins that are known to interact with tmRNA were purified, and their interactions with the defective RNA variants were examined in vitro. Based on phylogenetic and functional data, an additional structural motif consisting of a quartet composed of non-Watson–Crick base pairs 5′-YGAC-3′:5′-GGAC-3′ involving some of the conserved nucleotides next to the tRNA-like portion is proposed. Overall, the highly conserved nucleotides around the tRNA-like portion are maintained for both structural and functional requirements during evolution.     

Five pseudoknots are present at the 204 nucleotides long 3'' noncoding region of tobacco mosaic virus RNA.

The 104 nucleotides long 3' terminal region of TMV RNA was shown previously to contain two pseudoknotted structures (Rietveld et al. (1984), EMBO J. 3, 2613-2619). We here present evidence for the occurrence, within the 204 nucleotides long 3' noncoding region, of another highly structured domain located immediately adjacent to the tRNA-like structure of 95 nucleotides (Joshi et al. (1985) Nucleic Acids Res. 13, 347-354). A model for the three-dimensional folding of this region, containing three more pseudoknots, is proposed on the basis of chemical modification and enzymatic digestion. The existence of these three consecutive pseudoknots was supported by sequence comparisons with the RNA from the related tobamoviruses TMV-L, CcTMV and CGMMV. Coaxial stacking of the six double helical segments involved gives rise to the formation of a 25 basepair long quasi-continuous double helix. The results show that the three-dimensional folding of the 3' non-translated region of tobamoviral RNAs is largely maintained by the formation of five pseudoknots. The organisation of this region in the RNA of the tobamovirus CcTMV suggests that recombinational events among aminoacylatable plant viral RNAs have to be considered.     

Fluorescence characterization of the transfer RNA-like domain of transfer messenger RNA in complex with small binding protein B

Transfer messenger RNA (tmRNA) and small binding protein B (SmpB) are the main components of the trans-translation rescue machinery that releases stalled ribosomes from defective mRNAs. Little is known about how SmpB binding affects the conformation of the tRNA-like domain (TLD) of tmRNA. It has been previously hypothesized that the absence of a D stem in the TLD provides flexibility in the elbow region of tmRNA, which can be stabilized by its interaction with SmpB. Here, we have used F?rster resonance energy transfer to characterize the global structure of the tRNA-like domain of tmRNA in the presence and absence of SmpB and as a function of Mg(2+) concentration. Our results show tight and specific binding of SmpB to tmRNA. Surprisingly, our data show that the global conformation and flexibility of tmRNA do not change upon SmpB binding. However, Mg(2+) ions induce an 11 ? compaction in the tmRNA structure, suggesting that the flexibility in the H2a stem may allow different conformations of tmRNA as the TLD and mRNA-like domain need to be positioned differently while moving through the ribosome.     

Functional significance of an evolutionarily conserved alanine (GCA) resume codon in tmRNA in Escherichia coli

Occasionally, ribosomes stall on mRNAs prior to the completion of the polypeptide chain. In Escherichia coli and other eubacteria, tmRNA-mediated trans-translation is a major mechanism that recycles the stalled ribosomes. The tmRNA possesses a tRNA-like domain and a short mRNA region encoding a short peptide (ANDENYALAA in E. coli) followed by a termination codon. The first amino acid (Ala) of this peptide encoded by the resume codon (GCN) is highly conserved in tmRNAs in different species. However, reasons for the high evolutionary conservation of the resume codon identity have remained unclear. In this study, we show that changing the E. coli tmRNA resume codon to other efficiently translatable codons retains efficient functioning of the tmRNA. However, when the resume codon was replaced with the low-usage codons, its function was adversely affected. Interestingly, expression of tRNAs decoding the low-usage codon from plasmid-borne gene copies restored efficient utilization of tmRNA. We discuss why in E. coli, the GCA (Ala) is one of the best codons and why all codons in the short mRNA of the tmRNA are decoded by the abundant tRNAs.     

Visualizing the transfer-messenger RNA as the ribosome resumes translation

Bacterial ribosomes stalled by truncated mRNAs are rescued by transfer-messenger RNA (tmRNA), a dual-function molecule that contains a tRNA-like domain (TLD) and an internal open reading frame (ORF). Occupying the empty A site with its TLD, the tmRNA enters the ribosome with the help of elongation factor Tu and a protein factor called small protein B (SmpB), and switches the translation to its own ORF. In this study, using cryo-electron microscopy, we obtained the first structure of an in vivo-formed complex containing ribosome and the tmRNA at the point where the TLD is accommodated into the ribosomal P site. We show that tmRNA maintains a stable 'arc and fork' structure on the ribosome when its TLD moves to the ribosomal P site and translation resumes on its ORF. Based on the density map, we built an atomic model, which suggests that SmpB interacts with the five nucleotides immediately upstream of the resume codon, thereby determining the correct selection of the reading frame on the ORF of tmRNA.     

Phylogenetic analysis of tmRNA secondary structure.

The bacterial tmRNA acts with dual tRNA-like and mRNA-like character to tag incomplete translation products for degradation. Comparative analysis of 17 tmRNA genes (including eight new sequences) has allowed us to deduce conserved features of the tmRNA secondary structure. Except in a segment that includes the first codon of the tag reading frame, tmRNA is highly structured, with four pseudoknots and a total of 11 conserved base pairing regions. The previously identified tRNA minihelix structure is connected by a long base paired region to a large structured domain composed of a pseudoknot, followed by the tag reading frame and a string of three rather similar pseudoknots. The conservation of numerous structural elements among diverse eubacterial species indicates that these elements have important function beyond simply forming an endonuclease-resistant link between the reading frame and the tRNA-like domain.     

Functional and structural analysis of a pseudoknot upstream of the tag-encoded sequence in E. coli tmRNA

Escherichia coli tmRNA (transfer-messenger RNA) facilitates a trans-translation reaction in which a stalled ribosome on a terminatorless mRNA switches to an internal coding sequence in tmRNA, resulting in the addition of an 11 amino acid residue tag to the truncated protein that is a signal for degradation and in recycling of the stalled ribosome. A tmRNA secondary structure model with a partial tRNA-like structure and several pseudoknots was recently proposed. This report describes an extensive mutational analysis of one predicted pseudoknot (PK1) located upstream of the E. coli tmRNA tag-encoded sequence. Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all the engineered RNA variants. To characterize structure-function relationships for the tmRNA mutants, their solution conformations were investigated by using structural probes and by measuring the temperature dependence of their UV absorbance. This analysis strongly supports the presence of a pseudoknot in E. coli tmRNA, and its involvement in trans-translation. Mutations disrupting the first stem of the pseudoknot inactivate function and promote stable alternative conformations. Mutations of the second stem of the pseudoknot also effect both functions. The nucleotide stretch between the two stems (loop 2) is required for efficient trans-translation, and nucleotides at positions 61 and 62 must be guanine residues. The probing data suggest the presence of magnesium ion(s) interacting with loop 2. The loops crossing the minor and major grooves can be mutated without significant effects on tmRNA function. Nucleotide insertion or deletion between the pseudoknot and the coding sequence do not change the mRNA frame of the tag-peptide sequence, suggesting that the pseudoknot structure is not a determinant for the resumption of translation.     

Contributions of pseudoknots and protein SmpB to the structure and function of tmRNA in trans-translation

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 trans-translation. Taken together, these results provide unanticipated insights into the functions of the pseudoknots and protein SmpB during tmRNA folding, maturation, and protein synthesis.     

SmpB functions in various steps of trans-translation

tmRNA has a dual function as a tRNA and an mRNA to facilitate trans-translation, in which a ribosome can switch between translation of a truncated mRNA and the tmRNA’s tag sequence. SmpB is a tmRNA binding protein that has been identified to be essential for trans-translation in vivo. To further study the function of SmpB, an S30 fraction from an Escherichia coli strain, in which the set of genes for SmpB and tmRNA has been deleted from the genome, and His-tagged SmpB active in trans-translation were prepared. The SmpB-depleted S30 fraction had an ability to facilitate poly(U)-dependent tag-peptide synthesis in vitro when purified His-tagged SmpB was exogenously added together with tmRNA, although SmpB was not required for in vitro poly(U)-dependent poly(Phe) synthesis. It was also found that depletion of SmpB leads to a decrease in the level of tmRNA in the cell. In addition, SmpB considerably enhanced the aminoacylation of tmRNA by alanyl-tRNA synthetase in vitro. The aminoacylation enhancement by SmpB, the binding of SmpB to tmRNA and the effect of depletion of SmpB on the expression level of tmRNA in the cell were all affected by some mutations in the tRNA-like domain which cause a defect in ribosome binding leading to a trans-translation deficiency. These results demonstrate that, via binding to the tRNA-like domain of tmRNA, SmpB plays various roles: rescuing the tmRNA molecule from degradation in the cell, enhancing the aminoacylation of tmRNA and mediating the binding of tmRNA to ribosome.     

Simultaneous and functional binding of SmpB and EF-Tu-TP to the alanyl acceptor arm of tmRNA.

Transfer-messenger RNA (tmRNA) mimics functions of aminoacyl-tRNA and mRNA, subsequently, when rescuing stalled ribosomes on a 3' truncated mRNA without stop codon in bacteria. In addition, this mechanism marks prematurely terminated proteins by a C-terminal peptide tag as a signal for degradation by specific cellular proteases. For Escherichia coli, previous studies on initial steps of this "trans-translation" mechanism revealed that tmRNA alanylation by Ala-tRNA synthetase and binding of Ala-tmRNA by EF-Tu-GTP for subsequent delivery to stalled ribosomes are inefficient when compared to analogous reactions with canonical tRNA(Ala). In other studies, protein SmpB and ribosomal protein S1 appeared to bind directly to tmRNA and to be indispensable for trans-translation. Here, we have searched for additional and synergistic effects of the latter two on tmRNA alanylation and its subsequent binding to EF-Tu-GTP. Kinetic analysis of functioning combined with band-shift experiments and structural probing demonstrate, that tmRNA may indeed form a multimeric complex with SmpB, S1 and EF-Tu-GTP, which leads to a considerably enhanced efficiency of the initial steps of trans-translation. Whereas S1 binds to the mRNA region of tmRNA, we have found that SmpB and EF-Tu both interact with its acceptor arm region. Interaction with SmpB and EF-Tu was also observed at the acceptor arm of Ala-tRNA(Ala), but there the alanylation efficiency was inhibited rather than stimulated by SmpB. Therefore, SmpB may function as an essential modulator of the tRNA-like acceptor arm of tmRNA during its successive steps in trans-translation.     

The 5'' splice site: phylogenetic evolution and variable geometry of association with U1RNA.

The 5' splice site sequences of 3294 introns from various organisms (1-672) were analyzed in order to determine the rules governing evolution of this sequence, which may shed light on the mechanism of cleavage at the exon-intron junction. The data indicate that, currently, in all organisms, a common sequence 1GUAAG6U and its derivatives are used as well as an additional sequence and its derivatives, which differ in metazoa (G/1GUgAG6U), lower eucaryotes (1GUAxG6U) and higher plants (AG/1GU3A). They all partly resemble the prototype sequence AG/1GUAAG6U whose 8 contigous nucleotides are complementary to the nucleotides 4-11 of U1RNA, which are perfectly conserved in the course of phylogenetic evolution. Detailed examination of the data shows that U1RNA can recognize different parts of 5' splice sites. As a rule, either prototype nucleotides at position -2 and -1 or at positions 4, 5 or 6 or at positions 3-4 are dispensable provided that the stability of the U1RNA-5' splice site hybrid is conserved. On the basis of frequency of sequences, the optimal size of the hybridizable region is 5-7 nucleotides. Thus, the cleavage at the exon-intron junction seems to imply, first, that the 5' splice site is recognized by U1RNA according to a "variable geometry" program; second, that the precise cleavage site is determined by the conserved sequence of U1RNA since it occurs exactly opposite to the junction between nucleotides C9 and C10 of U1RNA. The variable geometry of the U1RNA-5' splice site association provides flexibility to the system and allows diversification in the course of phylogenetic evolution.