Activity of Euglena gracilis chloroplast ribosomes with procaryotic and eucaryotic initiation factors

A method that permits the preparation of Euglena gracilis chloroplast 30 S ribosomal subunits that are largely free of endogenous initiation factors and that are active in the binding of fMet-tRNA in response to poly(A, U, G), has been developed. These 30 S subunits have been tested for activity in initiation complex formation with initiation factors from both procaryotes and eucaryotes. We have observed that Escherichia coli IF-2 binds fMet-tRNA nearly as well to Euglena chloroplast ribosomal subunits as it does to its homologous subunits. Neither wheat germ eIF-2 nor Euglena eIF-2A can bind fMet-tRNA efficiently to Euglena chloroplast or E. coli 30 S subunits although both are active with wheat germ 40 S ribosomal subunits. Euglena chloroplast 68 S ribosomes will also bind the initiator tRNA. Both E. coli IF-2 and E. coli IF-3 stimulate this reaction on chloroplast ribosomes with approximately the same efficiency as they do on their homologous ribosomes. E. coli IF-1 enhances the binding of fMet-tRNA to the chloroplast 68 S ribosomes when either IF-2 or IF-3 is limiting. The chloroplast ribosomes unlike E. coli ribosomes show considerable activity over a broad range of Mg2+ ion concentrations.     

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.     

Immunological characterization of the complex forms of chloroplast translational initiation factor 2 from Euglena gracilis.

Euglena gracilis chloroplast translational initiation factor 2 (IF-2chl) occurs in several complex forms ranging in molecular mass from 200 to 800 kDa. Subunits of 97 to greater than 200 kDa have been observed in these preparations. Two monoclonal antibodies were prepared against the 97-kDa subunits of IF-2chl. Both of these antibodies recognize all of the higher molecular mass forms of this factor, suggesting that these subunits are closely related. Gel filtration chromatography indicates that the higher molecular mass subunits of IF-2chl are present in the higher molecular mass complexes, whereas the smaller subunits are present in the 200-400 kDa forms of IF-2chl. Probing extracts of light-induced and dark-grown cells with the antibodies indicates that the light induction of this chloroplast factor results from the synthesis of new polypeptide rather than from the activation of an inactive precursor form of the protein. Both the higher and lower molecular mass subunits of IF-2chl are present in 30 S initiation complexes as indicated by Western analysis. The binding of IF-2chl to chloroplast 30 S ribosomal subunits requires the presence of GTP, but does not require fMet-tRNA, messenger RNA, or other initiation factors. Neither polyclonal nor monoclonal antibodies against E. gracilis IF-2chl cross-react with Escherichia coli IF-2 or with animal mitochondrial IF-2.     

Alternative occupancy of a dual ribosomal binding site by mRNA affected by translation initiation factors

The interaction between Escherichia coli 30S ribosomal subunits and mRNAs, and the effect of the initiation factors on this process, have been studied using MS2 RNA, polyribonucleotides and model mRNAs encoded by synthetic genes. The interactions were analyzed by gel filtration, by sucrose gradient centrifugation and by competition for ribosome binding between the various mRNAs and a Shine-Dalgarno deoxyoctanucleotide. It was found that the initiation factors do not significantly affect the Shine-Dalgarno interaction nor the apparent Ka values of the 30S-subunit-mRNA binary complexes, but influence the positioning of the mRNAs on the 30S subunit with respect to the Shine-Dalgarno octanucleotide. The results suggest that, in the absence of initiation factors, the mRNA occupies a ribosomal "stand-by" site which is close to or includes the region where the Shine-Dalgarno interaction takes place; in the presence of the factors, the mRNA is shifted away from the stand-by site, towards another ribosomal site with similar affinity for the mRNA. This shift does not require the presence of fMet-tRNA and, depending upon the type of mRNA, is mediated by IF-2 and/or IF-3.     

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.     

Initiation of Escherichia coli ribosomes on matrix coupled mRNAs studied by optical biosensor technique

The optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, has been used to study the initiation of protein synthesis by E. coli ribosomes on surface coupled mRNA. mRNA was first periodate oxidized and then hydrazide coupled to the surface of a CM5 sensor chip. The formation of initiation complexes on the surface coupled mRNA was monitored in real-time with a BIACORE 2000 instrument. Mature 70S*mRNA*fMet-tRNA(Met) initiation complexes were assembled on mRNA by sequential introduction of the 30S and 50S subunits supplemented with appropriate initiation factors and fMet-tRNA(Met). We show that the formation of 70S*mRNA complexes on the surface coupled mRNA proceeds efficiently only in the presence of tRNA. Moreover, 70S*mRNA*fMet-tRNA(Met) complexes formed with fMet-tRNA(Met) are more stable than similar complexes formed with deacylated tRNAs. The efficient formation and slow dissociation of mature 70S*mRNA*fMet-tRNA(Met) initiation complexes are most easily explained by the stabilization of the interaction of the ribosomal subunits by fMet-tRNA(Met). This work demonstrates the feasibility of the BIACORE technique for studying the initiation of protein synthesis.     

Free initiation factors eIF4A and eIF4B are dispensable for translation initiation on uncapped mRNAs

The formation of ribosomal 48S initiation complexes at the start AUG codon of uncapped mRNA leader sequences was studied using the methodology of primer extension inhibition (toe-printing). The experiments were performed in the system composed of purified individual components required for translation initiation. The formation of ribosomal 48S initiation complexes at the initiation codon was tested depending on the presence of the initiation factors eIF4F, eIF4A, and eIF4B. Several mRNAs containing short leader sequences lacking the extended secondary structure were studied. It was found that 48S ribosomal complexes at mRNAs with such leaders were not formed in the absence of eIF4F. In contrast, the removal of either eIF4A or eIF4B from the experimental system was found to be dispensable for the formation of the 48S complex.     

Depletion of free 30S ribosomal subunits in Escherichia coli by expression of RNA containing Shine-Dalgarno-like sequences.

We have constructed synthetic coding sequences for the expression of poly(alpha,L-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in Escherichia coli. These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5'-GAGGAGG-3') that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs. An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA. Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs. Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin. The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA. We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA.     

Development and characterization of a reconstituted yeast translation initiation system

To provide a bridge between in vivo and in vitro studies of eukaryotic translation initiation, we have developed a reconstituted translation initiation system using components from the yeast Saccharomyces cerevisiae. We have purified a minimal set of initiation factors (elFs) that, together with yeast 80S ribosomes, GTP, and initiator methionyl-tRNA, are sufficient to assemble active initiation complexes on a minimal mRNA template. The kinetics of various steps in the pathway of initiation complex assembly and the formation of the first peptide bond in vitro have been explored. The formation of active initiation complexes in this system is dependent on ribosomes, mRNA, Met-tRNAi, GTP hydrolysis, elF1, elF1A, elF2, elF5, and elF5B. Our data indicate that elF1 and elF1A both facilitate the binding of the elF2 x GTP x Met-tRNAi complex to the 40S ribosomal subunit to form the 43S complex. elF5 stimulates a step after 43S complex formation, consistent with its proposed role in activating GTP hydrolysis by elF2 upon initiation codon recognition. The presence of elF5B is required for the joining of the 40S and 60S subunits to form the 80S initiation complex. The step at which each of these factors acts in this reconstituted system is in agreement with previous data from in vivo studies and work using reconstituted mammalian systems, indicating that the system recapitulates fundamental events in translation initiation in eukaryotic cells. This system should allow us to couple powerful yeast genetic and molecular biological experiments with in vitro kinetic and biophysical experiments, yielding a better understanding of the molecular mechanics of this central, complex process.     

Modulation of ribosomal recruitment to 5'-terminal start codons by translation initiation factors IF2 and IF3

Sequence determinants and structural features of the RNA govern mRNA-ribosome interaction in bacteria. However, ribosomal recruitment to leaderless mRNAs, which start directly with the AUG start codon and do not bear a Shine-Dalgarno sequence like canonical mRNAs, does not appear to rely on 16S rRNA-mRNA interactions. Here, we have studied the effects of translation initiation factors IF2 and IF3 on 30S initiation at a 5'-terminal AUG and at a competing downstream canonical ribosome binding site. We show that IF2 affects the forward kinetics of 30S initiation complex formation at the 5'-terminal AUG as well as the stability of these complexes. Moreover, the IF2:IF3 molar ratio was found to play a decisive role in translation initiation of a leaderless mRNA both in vitro and in vivo indicating that the translational efficiency of an mRNA is not only intrinsically determined but can be altered depending on the availability of components of the translational machinery.     

Escherichia coli 30S mutants lacking protein S20 are defective in translation initiation

The 30S ribosomal subunits derived from Escherichia coli TA114, a a temperature-sensitive mutant lacking ribosomal protein S20, were shown to be defective in two ways: (a) they have a reduced capacity for association with the 50S ribosomal subunit which results in the impairment of most of the functions requiring a coordinated interaction between the two subunits; (b) they are defective in functions which do not require their interaction with the large subunit (i.e., the formation of ternary complexes with aminocyl-tRNAs and templates, including the formation of 30S initiation complexes with fMet-tRNA and mRNA). The 30S (-S20) subunits seem to interact normally with both template and aminoacyl-tRNA individually, but appear to be impaired in the rate-limiting isomerization step leading to the formation of a codon-anticodon interaction in the P site.     

Isolation of Euglena gracilis chloroplast 5S ribosomal RNA and mapping the 5S rRNA gene on chloroplast DNA.

Ribosomal RNA (5S) from Euglena gracilis chloroplasts was isolated by preparative electrophoresis, labeled in vitro with 125I, and hybridized to restriction nuclease fragments from chloroplast DNA or cloned chloroplast DNA segments. Euglena chloroplast 5S rRNA is encoded in the chloroplast genome. The coding region of 5S rRNA has been positioned within the 5.6 kilobase pair (kbp) repeat which also codes for 16S and 23S rRNA. There are three 5S rRNA genes on the 130-kbp genome. The order of RNAs within a single repeat is 16S-23S-5S. The organization and size of the Euglena chloroplast ribosomal repeat is very similar to the ribosomal RNA operons of Escherichia coli.     

Identification and characterization of large, complex forms of chloroplast translational initiation factor 2 from Euglena gracilis

Chromatography of partially purified preparations of Euglena gracilis chloroplast initiation factor 2 (IF-2chl) on gel filtration resins indicates that this factor is present in high molecular mass forms ranging from 200 to 700 kDa. The higher molecular weight complexes can be separated from the 200,000 Mr form of this factor by chromatography on DEAE-cellulose. Further purification indicates that the majority of the IF-2chl is present as dimeric, tetrameric, and probably hexameric complexes of polypeptides of 97,000-110,000 in molecular weight. In addition, one form consisting of subunits of about 200,000 Mr has been detected. All of these species are active in promoting fMet-tRNA binding to chloroplast 30 S subunits in a message-dependent reaction. Initiation complex formation promoted by IF-2chl requires the presence of GTP. Similar levels of binding are obtained when GTP is replaced by a nonhydrolyzable analog suggesting that IF-2chl is acting stoichiometrically rather than catalytically under the conditions used. The activity of this factor is stimulated by the presence of either Escherichia coli or chloroplast IF-3. None of the forms of IF-2chl detected is active on E. coli ribosomes.     

Euglena gracilis chloroplast initiation factor 2. Identification and initial characterization

The chloroplast protein synthesis factor responsible for the binding of fMet-tRNAMeti to chloroplast 30 S ribosomal subunits (IF-2chl) has been identified in whole cell extracts of Euglena gracilis. The IF-2chl activity is present in considerably higher amounts in extracts of light-grown cells than in extracts of dark-grown cells. About 90% of this activity is found in the postribosomal supernatant of the cell. Chromatography on phosphocellulose results in the partial purification of IF-2chl and separates the chloroplast factor from the cytoplasmic factor eIF-2A. The binding of fMet-tRNAMeti to chloroplast 30 S subunits is message-dependent as observed for prokaryotic systems. In addition, GTP stimulates the IF-2chl-dependent reaction 3-fold. The binding reaction shows broad monovalent and divalent cation optima. The activity of IF-2chl is stimulated 2-fold by the addition of either Escherichia coli IF-1 or IF-3, and 4-fold by the inclusion of both factors. Chloroplast IF-2 is quite active on the homologous 30 S ribosomal subunits but shows little activity on E. coli 30 S or wheat germ 40 S subunits.     

Structured RNA upstream of insect cap distal iron responsive elements enhances iron regulatory protein-mediated control of translation

Iron regulatory protein (IRP) blocks ribosomal assembly by binding to an iron responsive element (IRE) located proximal (<60 nts) to the mRNA cap, thereby repressing translation. Constructs with IREs located 60–100 nts from the cap permit ribosomal assembly but the ribosomes pause at IRE/IRP complexes resulting in partial repression of translation. However, insect ferritin mRNAs have cap-distal IREs located 90–156 nts from the cap. Because iron can be toxic, it seems unlikely that insects would be unable to fully regulate ferritin synthesis at the level of translation. Calpodes ferritin consists of two subunits, S and G. In vitro translation of Calpodes ferritin and IRP1 from fat body mRNA yields only G subunits suggesting that IRP1 more efficiently represses translation of the S subunit than the G. When repression is removed by the addition of IRE competitor RNA, the synthesis of both subunits is greatly increased. S and G ferritin mRNAs have identical IREs in similar far cap-distal positions. While both ferritin mRNAs are predicted to have stem-loops between the IRE and the RNA cap, in general insect S mRNAs have more cap-proximal RNA structure than G mRNAs. Therefore, we examined the effect of upstream secondary structure on ribosomal assembly onto S ferritin mRNA constructs using sucrose gradient analysis of translation initiation complexes. We found no evidence for ribosomal assembly on wild type Calpodes S ferritin mRNA in the presence of IRP1 while constructs lacking the wild type secondary structure showed ribosomal pausing. Constructs with wild type secondary structure preceded by an unstructured upstream leader assemble ribosomes in the presence or absence of IRP1. Sequence and RNA folding analyses of other insect ferritins with cap-distal IREs failed to identify any common sequences or IRE-like structures that might bind to IRP1 with lower affinity or to another RNA binding protein. We propose that stem-loops upstream from the IRE act like pleats that shorten the effective distance between the IRE and cap and allow full translational repression by IRP1. In this way some cap-distal IREs may function like cap-proximal ones.     

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.     

From stand-by to decoding site. Adjustment of the mRNA on the 30S ribosomal subunit under the influence of the initiation factors.

The hypothesis of an adjustment of the mRNA in its ribosomal channel under the influence of the initiation factors has been tested by site-directed crosslinking experiments. Complexes containing 30S subunits with bound mRNA having 4-thio-uracil at specific positions were prepared in the presence or absence of initiation factors and/or fMet-tRNA and subjected to UV irradiation to obtain specific crosslinks of the radioactively labeled mRNA with bases of the 16S rRNA and with ribosomal proteins. The subsequent identification of the specific sites of both mRNA and rRNA and individual ribosomal proteins involved in the crosslinking, obtained under different conditions of complex formation, provide direct evidence for the occurrence of a partial relocation of the mRNA on the 30S ribosomal subunits under the influence of the factors. The nature of this mRNA relocation is compatible with our previous proposal of a shift of the template from an initial ribosomal "stand-by site" to a second site closer to that occupied when the initiation triplet of the mRNA is decoded in the P-site.     

Chloroplast initiation factor 3 from Euglena gracilis. Identification and initial characterization

A chloroplast ribosome dissociation factor (IF-3chl) has been identified in whole cell extracts of Euglena gracilis. This work represents the first report of an organellar ribosome dissociation factor. E. gracilis IF-3chl facilitates the dissociation of Escherichia coli ribosomes as demonstrated by sucrose density gradient analysis. Chloroplast IF-3 stimulates initiation complex formation on E. coli ribosomes with natural mRNA from the bacteriophage MS2. In addition, IF-3chl is effective in initiation complex formation with Euglena chloroplast or E. coli ribosomes in the presence of synthetic mRNA. IF-3chl is induced 12-fold by exposure of the cells to light. The chloroplast factor has been purified 30-fold by chromatography on DEAE-cellulose and phosphocellulose. The chromatographic properties of this factor differ considerably from those of prokaryotic ribosome dissociation factors.     

Interaction of the Escherichia coli fdhF mRNA hairpin promoting selenocysteine incorporation with the ribosome.

The codon UGA located 5' adjacent to an mRNA hairpin within fdhF mRNA promotes the incorporation of the amino acid selenocysteine into formate dehydrogenase H of Escherichia coli. The loop region of this mRNA hairpin has been shown to bind to the special elongation factor SELB, which also forms a complex with selenocysteinyl-tRNA(Sec) and GTP. We designed seven different mRNA constructs derived from the fdhF mRNA which contain a translation initiation region including an AUG initiation codon followed by no, one, two, three, four, five or six UUC phenylalanine codon(s) and the UGA selenocysteine codon 5' adjacent to the fdhF mRNA hairpin. By binding these different mRNA constructs to 30S ribosomal subunits in vitro we attempted to mimic intermediate steps of elongation of a structured mRNA approaching the ribosome by one codon at a time. Toeprint analysis of the mRNA-ribosome complexes showed that the presence of the fdhF mRNA hairpin strongly interferes with binding of the fdhF mRNA to 30S ribosomal subunits as soon as the hairpin is placed closer than 16 bases to the ribosomal P-site. Binding is reduced up to 25-fold compared with mRNA constructs where the hairpin is located outside the ribosomal mRNA track. Surprisingly, no toeprint signals were observed in any of our mRNA constructs when tRNA(Sec) was used instead of tRNA(fMet). Lack of binding of selenocysteinyl-tRNA(Sec) to the UGA codon was attributed to steric hindrance by the fdhF mRNA hairpin. By chemical probing of the shortest mRNA construct (AUG-UGA-fdhF hairpin) bound to 30S ribosomal subunits we demonstrate that the hairpin structure is not unfolded in the presence of ribosomes in vitro; also, this mRNA is not translated in vivo when fused in-frame 5' of the lacZ gene. Therefore, our data indicate that the fdhF mRNA hairpin has to be unfolded during elongation prior to entering the ribosomal mRNA track and we propose that the SELB binding domain within the fdhF mRNA is located outside the ribosomal mRNA track during decoding of the UGA selenocysteine codon by the SELB-selenocysteinyl-tRNA(Sec)-GTP complex.