Differential inhibition of 30S and 70S translation initiation complexes on leaderless mRNA by kasugamycin

In contrast to canonical mRNAs, translation of leaderless mRNA has been previously reported to continue in the presence of the antibiotic kasugamycin. Here, we have studied the effect of the antibiotic on determinants known to affect translation of leadered and leaderless mRNAs. Kasugamycin did not affect the Shine-Dalgarno (SD)-anti-SD (aSD) interaction or the function of translation initiation factor 3 (IF3). Thus, the preferential translation of leaderless mRNA in the presence of kasugamycin can neither be attributed to an expanding pool of 30S subunits with a "blocked" aSD nor to a lack of action of IF3, which has been shown to discriminate against translation initiation at 5'-terminal start codons. Using toeprinting, we observed that on leaderless mRNA 70S in contrast to 30S translation initiation complexes are comparatively resistant to the antibiotic. These results taken together with the known preference of 70S ribosomes for 5'-terminal AUGs lend support to the hypothesis that translation of leaderless mRNAs may as well proceed via an alternative initiation pathway accomplished by intact 70S ribosomes.     

Evidence for the translation initiation of leaderless mRNAs by the intact 70 S ribosome without its dissociation into subunits in eubacteria

In eubacteria, the dissociation of the 70 S ribosome into the 30 S and 50 S subunits is the essential first step for the translation initiation of canonical mRNAs that possess 5'-leader sequences. However, a number of leaderless mRNAs that start with the initiation codon have been identified in some eubacteria. These have been shown to be translated efficiently in vivo. Here we investigated the process by which leaderless mRNA translation is initiated by using a highly reconstituted cell-free translation system from Escherichia coli. We found that leaderless mRNAs bind preferentially to 70 S ribosomes and that the leaderless mRNA.70 S.fMet-tRNA complex can transit from the initiation to the elongation phase even in the absence of initiation factors (IFs). Moreover, leaderless mRNA translation proceeds more efficiently if the intact 70 S ribosome is involved compared with the 30 S subunit. Furthermore, excess amounts of IF3 inhibit leaderless mRNA translation, probably because it promotes the disassembly of the 70 S ribosome into subunits. Finally, excess amounts of fMet-tRNA facilitate the IF-independent translation of leaderless mRNA. These observations strongly suggest that leaderless mRNA translation is initiated by the assembled 70 S ribosome and thereby bypasses the dissociation process.     

A leaderless mRNA can bind to mammalian 80S ribosomes and direct polypeptide synthesis in the absence of translation initiation factors

Translation initiation in eukaryotic cells is known to be a complex multistep process which involves numerous protein factors. Here we demonstrate that leaderless mRNAs with initiator Met-tRNA can bind directly to 80S mammalian ribosomes in the absence of initiation factors and that the complexes thus formed are fully competent for the subsequent steps of polypeptide synthesis. We show that the canonical 48S pathway of eukaryotic translation initiation has no obvious advantage over the 80S pathway of translation initiation on leaderless mRNAs and suggest that, in the presence of competing mRNAs containing a leader, the latter mechanism will be preferred. The direct binding of the leaderless mRNA to the 80S ribosome was precluded when such an mRNA was supplied with a 5' leader, irrespective of whether it was in a totally single-stranded conformation or was prone to base pairing. The striking similarity between the mechanisms of binding of leaderless mRNAs with mammalian 80S or bacterial 70S ribosomes gives support to the idea that the alternative mode of translation initiation used by leaderless mRNAs represents a relic from early steps in the evolution of the translation apparatus.     

Isolation and characterization of ribosomes and translation initiation factors from the gram-positive soil bacterium Streptomyces lividans

A primer extension inhibition (toeprint) assay was developed using ribosomes and ribosomal subunits from Streptomyces lividans. This assay allowed the study of ribosome binding to streptomycete leaderless and leadered mRNA. Purified 30S subunits were unable to form a ternary complex on aph leaderless mRNA, whereas 70S ribosomes could form ternary complexes on this mRNA. 30S subunits formed ternary complexes on leadered aph and malE mRNA. The translation initiation factors (IF1, IF2, and IF3) from S. lividans were isolated and included in toeprint and filter binding assays with leadered and leaderless mRNA. Generally, the IFs reduced the toeprint signal on leadered mRNA; however, incubation of IF1 and IF2 with 30S subunits that had been washed under high-salt conditions promoted the formation of a ternary complex on aph leaderless mRNA. Our data suggest that, as reported for Escherichia coli, initiation complexes with leaderless mRNAs might use a novel pathway involving 70S ribosomes or 30S subunits bound by IF1 and IF2 but not IF3. Some mRNA-ribosome-initiator tRNA reactions that yielded weak or no toeprint signals still formed complexes in filter binding assays, suggesting the occurrence of interactions that are not stable in the toeprint assay.     

Ribosomes bind leaderless mRNA in Escherichia coli through recognition of their 5'-terminal AUG

Leaderless mRNAs are translated in the absence of upstream signals that normally contribute to ribosome binding and translation efficiency. In order to identify ribosomal components that interact with leaderless mRNA, a fragment of leaderless cI mRNA from bacteriophage λ, with a 4-thiouridine (4^S-U) substituted at the +2 position of the AUG start codon, was used to form cross-links to Escherichia coli ribosomes during binary (mRNA+ribosome) and ternary (mRNA+ribosome+initiator tRNA) complex formation. Ribosome binding assays (i.e., toeprints) demonstrated tRNA-dependent binding of leaderless mRNA to ribosomes; however, cross-links between the start codon and 30S subunit rRNA and r-proteins formed independent of initiator tRNA. Toeprints revealed that a leaderless mRNA's 5′-AUG is required for stable binding. Furthermore, the addition of a 5′-terminal AUG triplet to a random RNA fragment can make it both competent and competitive for ribosome binding, suggesting that a leaderless mRNA's start codon is a major feature for ribosome interaction. Cross-linking assays indicate that a subset of 30S subunit r-proteins, located at either end of the mRNA tunnel, contribute to tRNA-independent contacts and/or interactions with a leaderless mRNA's start codon. The interaction of leaderless mRNA with ribosomes may reveal features of mRNA binding and AUG recognition that are distinct from known signals but are important for translation initiation of all mRNAs.     

Leaderless mRNAs bind 70S ribosomes more strongly than 30S ribosomal subunits in Escherichia coli

By primer extension inhibition assays, 70S ribosomes bound with higher affinity, or stability, than did 30S subunits to leaderless mRNAs containing AUG or GUG start codons. Addition of translation initiation factors affected ribosome binding to leaderless mRNAs. Our results suggest that translation of leaderless mRNAs might initiate through a pathway involving 70S ribosomes or 30S subunits lacking IF3.     

Translation affects YoeB and MazF messenger RNA interferase activities by different mechanisms

Prokaryotic toxin–antitoxin loci encode mRNA cleaving enzymes that inhibit translation. Two types are known: those that cleave mRNA codons at the ribosomal A site and those that cleave any RNA site specifically. RelE of Escherichia coli cleaves mRNA at the ribosomal A site in vivo and in vitro but does not cleave pure RNA in vitro. RelE exhibits an incomplete RNase fold that may explain why RelE requires its substrate mRNA to presented by the ribosome. In contrast, RelE homologue YoeB has a complete RNase fold and cleaves RNA independently of ribosomes in vitro. Here, we show that YoeB cleavage of mRNA is strictly dependent on translation of the mRNA in vivo. Non-translated model mRNAs were not cleaved whereas the corresponding wild-type mRNAs were cleaved efficiently. Model mRNAs carrying frameshift mutations exhibited a YoeB-mediated cleavage pattern consistent with the reading frameshift thus giving strong evidence that YoeB cleavage specificity was determined by the translational reading frame. In contrast, site-specific mRNA cleavage by MazF occurred independently of translation. In one case, translation seriously influenced MazF cleavage efficiency, thus solving a previous apparent paradox. We propose that translation enhances MazF-mediated cleavage of mRNA by destabilization of the mRNA secondary structure.     

Requirements for ribosomal protein S1 for translation initiation of mRNAs with and without a 5' leader sequence

It has previously been proposed that Escherichia coli ribosomal protein S1 is required for the translation of highly structured mRNAs. In this study, we have examined the influence of structural features at or near the start codon of different mRNAs. The requirement for ribosomal protein S1 for translation initiation was determined when (i) the ribosome-binding site (RBS) was either preceded by a 5' non-translated leader sequence; (ii) the RBS was located 5' proximal to a mRNA start codon; and (iii) the start codon was the 5' terminal codon as exemplified by leaderless mRNAs. In vitro translation studies revealed that the leaderless lambda cl mRNA is translated with Bacillus stearothermophilusribosomes, naturally lacking a ribosomal protein S1 homologue, whereas ompA mRNA containing a 5' leader is not. These studies have been verified by toeprinting with E. coli ribosomes depleted for S1. We have shown that S1 is required for ternary complex formation on ompA mRNA but not for leaderless mRNAs or for mRNAs in which the RBS is close to the 5' end.     

Selective stimulation of translation of leaderless mRNA by initiation factor 2: evolutionary implications for translation

Translation initiation in bacteria involves a stochastic binding mechanism in which the 30S ribosomal subunit first binds either to mRNA or to initiator tRNA, fMet-tRNA(f)(Met). Leaderless lambda cI mRNA did not form a binary complex with 30S ribosomes, which argues against the view that ribosomal recruitment signals other than a 5'-terminal start codon are essential for translation initiation of these mRNAs. We show that, in Escherichia coli, translation initiation factor 2 (IF2) selectively stimulates translation of lambda cI mRNA in vivo and in vitro. These experiments suggest that the start codon of leaderless mRNAs is recognized by a 30S-fMet-tRNA(f)(Met)-IF2 complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes. We further show that leaderless lambda cI mRNA is faithfully translated in vitro in both archaebacterial and eukaryotic translation systems. This suggests that translation of leaderless mRNAs reflects a fundamental capability of the translational apparatus of all three domains of life and lends support to the hypothesis that the translation initiation pathway is universally conserved.     

Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA

Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.     

Ribosome collisions and translation efficiency: optimization by codon usage and mRNA destabilization

Individual mRNAs are translated by multiple ribosomes that initiate translation with an interval of a few seconds. The ribosome speed is codon dependent, and ribosome queuing has been suggested to explain specific data for translation of some mRNAs in vivo. By modeling the stochastic translation process as a traffic problem, we here analyze conditions and consequences of collisions and queuing. The model allowed us to determine the on-rate (0.8 to 1.1 initiations/s) and the time (1 s) the preceding ribosome occludes initiation for Escherichia coli lacZ mRNA in vivo. We find that ribosome collisions and queues are inevitable consequences of a stochastic translation mechanism that reduce the translation efficiency substantially on natural mRNAs. The cells minimize collisions by having its mRNAs being unstable and by a highly selected codon usage in the start of the mRNA. The cost of mRNA breakdown is offset by the concomitant increase in translation efficiency.     

Dissecting eukaryotic translation and its control by ribosome density mapping

Translation of an mRNA is generally divided into three stages: initiation, elongation and termination. The relative rates of these steps determine both the number and position of ribosomes along the mRNA, but traditional velocity sedimentation assays for the translational status of mRNA determine only the number of bound ribosomes. We developed a procedure, termed Ribosome Density Mapping (RDM), that uses site-specific cleavage of polysomal mRNA followed by separation on a sucrose gradient and northern analysis, to determine the number of ribosomes associated with specified portions of a particular mRNA. This procedure allows us to test models for translation and its control, and to examine properties of individual steps of translation in vivo. We tested specific predictions from the current model for translational control of GCN4 expression in yeast and found that ribosomes were differentially associated with the uORFs elements and coding region under different growth conditions, consistent with this model. We also mapped ribosome density along the ORF of several mRNAs, to probe basic kinetic properties of translational steps in yeast. We found no detectable decline in ribosome density between the 5′ and 3′ ends of the ORFs, suggesting that the average processivity of elongation is very high. Conversely, there was no queue of ribosomes at the termination site, suggesting that termination is not very slow relative to elongation and initiation. Finally, the RDM results suggest that less frequent initiation of translation on mRNAs with longer ORFs is responsible for the inverse correlation between ORF length and ribosomal density that we observed in a global analysis of translation. These results provide new insights into eukaryotic translation in vivo.     

Escherichia coli Ribosomal Protein S1 Unfolds Structured mRNAs Onto the Ribosome for Active Translation Initiation

Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5′ untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA–protein or mRNA–ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5′ ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features.     

YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes

In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl–tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.     

GTP-independent tRNA Delivery to the Ribosomal P-site by a Novel Eukaryotic Translation Factor

During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here, we report the tRNA binding to the P-site of 40 S ribosomes by a novel GTP-independent factor eIF2D isolated from mammalian cells. The binding of tRNA_i^Met occurs after the AUG codon finds its position in the P-site of 40 S ribosomes, the situation that takes place during initiation complex formation on the hepatitis C virus internal ribosome entry site or on some other specific RNAs (leaderless mRNA and A-rich mRNAs with relaxed scanning dependence). Its activity in tRNA binding with 40 S subunits does not require the presence of the aminoacyl moiety. Moreover, the factor possesses the unique ability to deliver non-Met (elongator) tRNAs into the P-site of the 40 S subunit. The corresponding gene is found in all eukaryotes and includes an SUI1 domain present also in translation initiation factor eIF1. The versatility of translation initiation strategies in eukaryotes is discussed.     

Translation initiation factor 3 antagonizes authentic start codon selection on leaderless mRNAs

In this study, we have examined the influence of initiation factors on translation initiation of leaderless mRNAs whose 5'-terminal residues are the A of the AUG initiating codon. A 1:1 ratio of initiation factors to ribosomes abolished ternary complex formation at the authentic start codon of different leaderless mRNAs. Supporting this observation, in vitro translation assays using limiting ribosome concentrations with competing leaderless λ c I and Escherichia coli ompA mRNAs, the latter containing a canonical ribosome binding site, revealed reduced cI synthesis relative to OmpA in the presence of added initiation factors. Using in vitro toeprinting and in vitro translation assays, we show that this effect can be attributed to IF3. Moreover, in vivo studies revealed that the translational efficiency of a leaderless reporter gene is decreased with increased IF3 levels. These studies are corroborated by the observed increased translational efficiency of a leaderless reporter construct in an infC mutant strain unable to discriminate against non-standard start codons. These results suggest that, in the absence of a leader or a Shine–Dalgarno sequence, the function(s) of IF3 limits stable 30S ternary complex formation.     

A single mutation in the IF3 N-terminal domain perturbs the fidelity of translation initiation at three levels

Bacterial translation initiation factor 3 (IF3) is involved in the fidelity of translation initiation at several levels, including start-codon discrimination, mRNA translation, and initiator-tRNA selection. The IF3 C-terminal domain (CTD) is required for binding to the 30S ribosomal subunit. N-terminal domain (NTD) function is less certain, but likely contributes to initiation fidelity. Point mutations in either domain can decrease initiation fidelity, but C-terminal domain mutations may be indirect. Here, the Y75N substitution mutation in the NTD is examined in vitro and in vivo. IF3_Y75N protein binds 30S subunits normally, but is defective in start-codon discrimination, inhibition of initiation on leaderless mRNA, and initiator-tRNA selection, thereby establishing a direct role for the IF3 NTD in these initiation processes. A model illustrating how IF3 modulates an inherent function of the 30S subunit is discussed.