Dynamics of release factor recycling during translation termination in bacteria.

In bacteria, release of newly synthesized proteins from ribosomes during translation termination is catalyzed by class-I release factors (RFs) RF1 or RF2, reading UAA and UAG or UAA and UGA codons, respectively. Class-I RFs are recycled from the post-termination ribosome by a class-II RF, the GTPase RF3, which accelerates ribosome intersubunit rotation and class-I RF dissociation. How conformational states of the ribosome are coupled to the binding and dissociation of the RFs remains unclear and the importance of ribosome-catalyzed guanine nucleotide exchange on RF3 for RF3 recycling in vivo has been disputed. Here, we profile these molecular events using a single-molecule fluorescence assay to clarify the timings of RF3 binding and ribosome intersubunit rotation that trigger class-I RF dissociation, GTP hydrolysis, and RF3 dissociation. These findings in conjunction with quantitative modeling of intracellular termination flows reveal rapid ribosome-dependent guanine nucleotide exchange to be crucial for RF3 action in vivo.

Mechanistic insights into translation inhibition by aminoglycoside antibiotic arbekacin.

How aminoglycoside antibiotics limit bacterial growth and viability is not clearly understood. Here we employ fast kinetics to reveal the molecular mechanism of action of a clinically used, new-generation, semisynthetic aminoglycoside Arbekacin (ABK), which is designed to avoid enzyme-mediated deactivation common to other aminoglycosides. Our results portray complete picture of ABK inhibition of bacterial translation with precise quantitative characterizations. We find that ABK inhibits different steps of translation in nanomolar to micromolar concentrations by imparting pleotropic effects. ABK binding stalls elongating ribosomes to a state, which is unfavorable for EF-G binding. This prolongs individual translocation step from ∼50 ms to at least 2 s; the mean time of translocation increases inversely with EF-G concentration. ABK also inhibits translation termination by obstructing RF1/RF2 binding to the ribosome. Furthermore, ABK decreases accuracy of mRNA decoding (UUC vs. CUC) by ∼80 000 fold, causing aberrant protein production. Importantly, translocation and termination events cannot be completely stopped even with high ABK concentration. Extrapolating our kinetic model of ABK action, we postulate that aminoglycosides impose bacteriostatic effect mainly by inhibiting translocation, while they become bactericidal in combination with decoding errors.

Estimation of peptide elongation times from ribosome profiling spectra.

Ribosome profiling spectra bear rich information on translation control and dynamics. Yet, due to technical biases in library generation, extracting quantitative measures of discrete translation events has remained elusive. Using maximum likelihood statistics and data set from Escherichia coli we develop a robust method for neutralizing technical biases (e.g. base specific RNase preferences in ribosome-protected mRNA fragments (RPF) generation), which allows for correct estimation of translation times at single codon resolution. Furthermore, we validated the method with available datasets from E. coli treated with antibiotic to inhibit isoleucyl-tRNA synthetase, and two datasets from Saccharomyces cerevisiae treated with two RNases with distinct cleavage signatures. We demonstrate that our approach accounts for RNase cleavage preferences and provides bias-corrected translation times estimates. Our approach provides a solution to the long-standing problem of extracting reliable information about peptide elongation times from highly noisy and technically biased ribosome profiling spectra.

Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions.

We studied the effects of aminoglycosides and changing Mg2+ ion concentration on the accuracy of initial codon selection by aminoacyl-tRNA in ternary complex with elongation factor Tu and GTP (T3) on mRNA programmed ribosomes. Aminoglycosides decrease the accuracy by changing the equilibrium constants of 'monitoring bases' A1492, A1493 and G530 in 16S rRNA in favor of their 'activated' state by large, aminoglycoside-specific factors, which are the same for cognate and near-cognate codons. Increasing Mg2+ concentration decreases the accuracy by slowing dissociation of T3 from its initial codon- and aminoglycoside-independent binding state on the ribosome. The distinct accuracy-corrupting mechanisms for aminoglycosides and Mg2+ ions prompted us to re-interpret previous biochemical experiments and functional implications of existing high resolution ribosome structures. We estimate the upper thermodynamic limit to the accuracy, the 'intrinsic selectivity' of the ribosome. We conclude that aminoglycosides do not alter the intrinsic selectivity but reduce the fraction of it that is expressed as the accuracy of initial selection. We suggest that induced fit increases the accuracy and speed of codon reading at unaltered intrinsic selectivity of the ribosome.

Complete kinetic mechanism for recycling of the bacterial ribosome.

How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of ∼5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.

Mechanism of fusidic acid inhibition of RRF- and EF-G-dependent splitting of the bacterial post-termination ribosome.

The antibiotic drug fusidic acid (FA) is commonly used in the clinic against gram-positive bacterial infections. FA targets ribosome-bound elongation factor G (EF-G), a translational GTPase that accelerates both messenger RNA (mRNA) translocation and ribosome recycling. How FA inhibits translocation was recently clarified, but FA inhibition of ribosome recycling by EF-G and ribosome recycling factor (RRF) has remained obscure. Here we use fast kinetics techniques to estimate mean times of ribosome splitting and the stoichiometry of GTP hydrolysis by EF-G at varying concentrations of FA, EF-G and RRF. These mean times together with previous data on uninhibited ribosome recycling were used to clarify the mechanism of FA inhibition of ribosome splitting. The biochemical data on FA inhibition of translocation and recycling were used to model the growth inhibitory effect of FA on bacterial populations. We conclude that FA inhibition of translocation provides the dominant cause of bacterial growth reduction, but that FA inhibition of ribosome recycling may contribute significantly to FA-induced expression of short regulatory open reading frames, like those involved in FA resistance.

Fusidic acid targets elongation factor G in several stages of translocation on the bacterial ribosome.

The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation (III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K50% ≈ 1 µm), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.

A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids.

There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNA(Ala)-based body (tRNA(AlaB)) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNA(PheB) body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNA(AlaB) body than from the tRNA(PheB) body. At ∼1 µM EF-Tu, tRNA(AlaB) conferred considerably faster incorporation kinetics than tRNA(PheB), especially in the case of the bulky bK. In contrast, the swap to the tRNA(AlaB) body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNA(AlaB) and tRNA(PheB) bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.

Optimal control of gene expression for fast proteome adaptation to environmental change.

Bacterial populations growing in a changing world must adjust their proteome composition in response to alterations in the environment. Rapid proteome responses to growth medium changes are expected to increase the average growth rate and fitness value of these populations. Little is known about the dynamics of proteome change, e.g., whether bacteria use optimal strategies of gene expression for rapid proteome adjustments and if there are lower bounds to the time of proteome adaptation in response to growth medium changes. To begin answering these types of questions, we modeled growing bacteria as stoichiometrically coupled networks of metabolic pathways. These are balanced during steady-state growth in a constant environment but are initially unbalanced after rapid medium shifts due to a shortage of enzymes required at higher concentrations in the new environment. We identified an optimal strategy for rapid proteome adjustment in the absence of protein degradation and found a lower bound to the time of proteome adaptation after medium shifts. This minimal time is determined by the ratio between the Kullback-Leibler distance from the pre- to the postshift proteome and the postshift steady-state growth rate. The dynamics of optimally controlled proteome adaptation has a simple analytical solution. We used detailed numerical modeling to demonstrate that realistic bacterial control systems can emulate this optimal strategy for rapid proteome adaptation. Our results may provide a conceptual link between the physiology and population genetics of growing bacteria.

Activation of initiation factor 2 by ligands and mutations for rapid docking of ribosomal subunits.

We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNA(i)). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNA(i), implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNA(i) and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10(-4) for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases.

pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA.

We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNA(fMet) to the aa-tRNAs Phe-tRNA(Phe), Ala-tRNA(Ala), Gly-tRNA(Gly), Pro-tRNA(Pro), Asn-tRNA(Asn), and Ile-tRNA(Ile), selected to cover a large range of intrinsic pK(a)-values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, pK(a)(obs), at which the rate was half maximal. The pK(a)(obs)-values were downshifted relative to the intrinsic pK(a)-value of aa-tRNAs in bulk solution. Gly-tRNA(Gly) had the smallest downshift, while Ile-tRNA(Ile) and Ala-tRNA(Ala) had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pK(a)-values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.

Antibiotic thermorubin tethers ribosomal subunits and impedes A-site interactions to perturb protein synthesis in bacteria.

Thermorubin (THB) is a long-known broad-spectrum ribosome-targeting antibiotic, but the molecular mechanism of its action was unclear. Here, our precise fast-kinetics assays in a reconstituted Escherichia coli translation system and 1.96 Å resolution cryo-EM structure of THB-bound 70S ribosome with mRNA and initiator tRNA, independently suggest that THB binding at the intersubunit bridge B2a near decoding center of the ribosome interferes with the binding of A-site substrates aminoacyl-tRNAs and class-I release factors, thereby inhibiting elongation and termination steps of bacterial translation. Furthermore, THB acts as an anti-dissociation agent that tethers the ribosomal subunits and blocks ribosome recycling, subsequently reducing the pool of active ribosomes. Our results show that THB does not inhibit translation initiation as proposed earlier and provide a complete mechanism of how THB perturbs bacterial protein synthesis. This in-depth characterization will hopefully spur efforts toward the design of THB analogs with improved solubility and effectivity against multidrug-resistant bacteria.

Inefficient delivery but fast peptide bond formation of unnatural L-aminoacyl-tRNAs in translation.

Translations with unnatural amino acids (AAs) are generally inefficient, and kinetic studies of their incorporations from transfer ribonucleic acids (tRNAs) are few. Here, the incorporations of small and large, non-N-alkylated, unnatural l-AAs into dipeptides were compared with those of natural AAs using quench-flow techniques. Surprisingly, all incorporations occurred in two phases: fast then slow, and the incorporations of unnatural AA-tRNAs proceeded with rates of fast and slow phases similar to those for natural Phe-tRNA(Phe). The slow phases were much more pronounced with unnatural AA-tRNAs, correlating with their known inefficient incorporations. Importantly, even for unnatural AA-tRNAs the fast phases could be made dominant by using high EF-Tu concentrations and/or lower reaction temperature, which may be generally useful for improving incorporations. Also, our observed effects of EF-Tu concentration on the fraction of the fast phase of incorporation enabled direct assay of the affinities of the AA-tRNAs for EF-Tu during translation. Our unmodified tRNA(Phe) derivative adaptor charged with a large unnatural AA, biotinyl-lysine, had a very low affinity for EF-Tu:GTP, while the small unnatural AAs on the same tRNA body had essentially the same affinities to EF-Tu:GTP as natural AAs on this tRNA, but still 2-fold less than natural Phe-tRNA(Phe). We conclude that the inefficiencies of unnatural AA-tRNA incorporations were caused by inefficient delivery to the ribosome by EF-Tu, not slow peptide bond formation on the ribosome.

Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting.

We demonstrate that ribosomes containing a messenger RNA (mRNA) with a strong Shine-Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant ribosomes are split by RRF/EF-G much more slowly than PTL ribosomes and by IF1/IF3 much more slowly than mRNA-containing ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of ribosome splitting by IF1/IF3 and by RRF/EF-G.

Slow peptide bond formation by proline and other N-alkylamino acids in translation.

Proteins are made from 19 aa and, curiously, one N-alkylamino acid ("imino acid"), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3-6 times slower than Phe incorporation from Phe-tRNA(Phe) depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNA(Pro) isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the "tRNA abundance" hypothesis.

Error-prone initiation factor 2 mutations reduce the fitness cost of antibiotic resistance.

Mutations in the fmt gene (encoding formyl methionine transferase) that eliminate formylation of initiator tRNA (Met-tRNA(i)) confer resistance to the novel antibiotic class of peptide deformylase inhibitors (PDFIs) while concomitantly reducing bacterial fitness. Here we show in Salmonella typhimurium that novel mutations in initiation factor 2 (IF2) located outside the initiator tRNA binding domain can partly restore fitness of fmt mutants without loss of antibiotic resistance. Analysis of initiation of protein synthesis in vitro showed that with non-formylated Met-tRNA(i) IF2 mutants initiated much faster than wild-type IF2, whereas with formylated fMet-tRNA(i) the initiation rates were similar. Moreover, the increase in initiation rates with Met-tRNA(i) conferred by IF2 mutations in vitro correlated well with the increase in growth rate conferred by the same mutations in vivo, suggesting that the mutations in IF2 compensate formylation deficiency by increasing the rate of in vivo initiation with Met-tRNA(i). IF2 mutants had also a high propensity for erroneous initiation with elongator tRNAs in vitro, which could account for their reduced fitness in vivo in a formylation-proficient strain. More generally, our results suggest that bacterial protein synthesis is mRNA-limited and that compensatory mutations in IF2 could increase the persistence of PDFI-resistant bacteria in clinical settings.

tmRNA.SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes.

Bacterial ribosomes stalled on faulty, often truncated, mRNAs lacking stop codons are rescued by trans-translation. It relies on an RNA molecule (tmRNA) capable of replacing the faulty mRNA with its own open reading frame (ORF). Translation of tmRNA ORF results in the tagging of faulty protein for degradation and its release from the ribosome. We used single-particle cryo-electron microscopy to visualize tmRNA together with its helper protein SmpB on the 70S Escherichia coli ribosome in states subsequent to GTP hydrolysis on elongation factor Tu (EF-Tu). Three-dimensional reconstruction and heterogeneity analysis resulted in a 15A resolution structure of the tmRNA.SmpB complex accommodated in the A site of the ribosome, which shows that SmpB mimics the anticodon- and D-stem of native tRNAs missing in the tRNA-like domain of tmRNA. We conclude that the tmRNA.SmpB complex accommodates in the ribosomal A site very much like an aminoacyl-tRNA during protein elongation.

Reiterative Synthesis by the Ribosome and Recognition of the N-Terminal Formyl Group by Biosynthetic Machinery Contribute to Evolutionary Conservation of the Length of Antibiotic Microcin C Peptide Precursor.

Microcin C (McC) is a peptide adenylate antibiotic produced by Escherichiacoli cells bearing a plasmid-borne mcc gene cluster. Most MccA precursors, encoded by validated mcc operons from diverse bacteria, are 7 amino acids long, but the significance of this precursor length conservation has remained unclear. Here, we created derivatives of E. colimcc operons encoding longer precursors and studied their synthesis and bioactivities. We found that increasing the precursor length to 11 amino acids and beyond strongly decreased antibiotic production. We found this decrease to depend on several parameters. First, reiterative synthesis of the MccA peptide by the ribosome was decreased at longer mccA open reading frames, leading to less efficient competition with other messenger RNAs. Second, the presence of a formyl group at the N-terminal methionine of the heptameric peptide had a strong stimulatory effect on adenylation by the MccB enzyme. No such formyl group stimulation was observed for longer peptides. Finally, the presence of the N-terminal formyl on the heptapeptide adenylate stimulated bioactivity, most likely at the uptake stage. Together, these factors should contribute to optimal activity of McC-like compounds as 7-amino-acid peptide moieties and suggest convergent evolution of several steps of the antibiotic biosynthesis pathway and their adjustment to sensitive cell uptake machinery to create a potent drug.IMPORTANCEEscherichia coli microcin C (McC) is a representative member of peptide-nucleotide antibiotics produced by diverse microorganisms. The vast majority of biosynthetic gene clusters responsible for McC-like compound production encode 7-amino-acid-long precursor peptides, which are C-terminally modified by dedicated biosynthetic enzymes with a nucleotide moiety to produce a bioactive compound. In contrast, the sequences of McC-like compound precursor peptides are not conserved. Here, we studied the consequences of E. coli McC precursor peptide length increase on antibiotic production and activity. We show that increasing the precursor peptide length strongly decreases McC production by affecting multiple biosynthetic steps, suggesting that the McC biosynthesis system has evolved under significant functional constraints to maintain the precursor peptide length.

Substrate-Induced Formation of Ribosomal Decoding Center for Accurate and Rapid Genetic Code Translation.

Accurate translation of genetic information is crucial for synthesis of functional proteins in all organisms. We use recent experimental data to discuss how induced fit affects accuracy of initial codon selection on the ribosome by aminoacyl transfer RNA in ternary complex ( T3) with elongation factor Tu (EF-Tu) and guanosine-5'-triphosphate (GTP). We define actual accuracy ([Formula: see text]) of a particular protein synthesis system as its current accuracy and the effective selectivity ([Formula: see text]) as [Formula: see text] in the limit of zero ribosomal binding affinity for T3. Intrinsic selectivity ([Formula: see text]), defined as the upper thermodynamic limit of [Formula: see text], is determined by the free energy difference between near-cognate and cognate T3 in the pre-GTP hydrolysis state on the ribosome. [Formula: see text] is much larger than [Formula: see text], suggesting the possibility of a considerable increase in [Formula: see text] and [Formula: see text] at negligible kinetic cost. Induced fit increases [Formula: see text] and [Formula: see text] without affecting [Formula: see text], and aminoglycoside antibiotics reduce [Formula: see text] and [Formula: see text] at unaltered [Formula: see text].

Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA.

In trans-translation transfer messenger RNA (tmRNA) and small protein B (SmpB) rescue ribosomes stalled on truncated or in other ways problematic mRNAs. SmpB promotes the binding of tmRNA to the ribosome but there is uncertainty about the number of participating SmpB molecules as well as their ribosomal location. Here, the interaction of SmpB with ribosomal subunits and ribosomes was studied by isolation of SmpB containing complexes followed by chemical modification of ribosomal RNA with dimethyl sulfate, kethoxal and hydroxyl radicals. The results show that SmpB binds 30S and 50S subunits with 1:1 molar ratios and the 70S ribosome with 2:1 molar ratio. SmpB-footprints are similar on subunits and the ribosome. In the 30S subunit, SmpB footprints nucleotides that are in the vicinity of the P-site facing the E-site, and in the 50S subunit SmpB footprints nucleotides that are located below the L7/L12 stalk in the 3D structure of the ribosome. Based on these results, we suggest a mechanism where two molecules of SmpB interact with tmRNA and the ribosome during trans-translation. The first SmpB molecule binds near the factor-binding site on the 50S subunit helping tmRNA accommodation on the ribosome, whereas the second SmpB molecule may functionally substitute for a missing anticodon stem-loop in tmRNA during later steps of trans-translation.