Mutagenesis at the mRNA decoding site in the 16S ribosomal RNA using the specialized ribosome system in Escherichia coli.

In the specialized ribosome system, a distinct pool of mutated ribosomes is dedicated to the translation of one particular mRNA species. This was accomplished by altering the Shine-Dalgarno sequence on the mRNA and its complementary anti-Shine-Dalgarno sequence on the plasmid-borne 16S rRNA gene. Here, using the specialized ribosome system, we were able to introduce mutations in key regions of the 16S rRNA and could study their effect on translation in vivo. The C1400 region has been implicated to play a role in the actual mRNA decoding process. Several ribosomal mutations were introduced in this region. We showed that substitution of the evolutionary highly conserved C1400 residue by a G- or an A-residue inhibits ribosomal activity by 80% and 50% respectively, whereas, a C to a U change at this conserved position does not affect overall ribosomal activity. The adjacent stem structure (1410-1490) was also examined. Disruption of the stem by replacing either one of the arms of this stem, with a different sequence, inhibits ribosomal activity by approximately 80%. A small but significant restoration of translation could be achieved by recreating a complementary stem with a different sequence. We found that full reversion of activity could be obtained when such mutated ribosomes were made spectinomycin resistant by introducing a C to A substitution at position 1192 which is located far away in the secondary structure map of the 16S rRNA molecule. Based on these results we conclude that some, but not all, of the nucleotides in the conserved C1400 region play a key role in translation.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro translation of poliovirus RNA: utilization of internal initiation sites in reticulocyte lysate.

The translation of poliovirus RNA in rabbit reticulocyte lysate was examined. Translation of poliovirus RNA in this cell-free system resulted in an electrophoretic profile of poliovirus-specific proteins distinct from that observed in vivo or after translation in poliovirus-infected HeLa cell extract. A group of proteins derived from the P3 region of the polyprotein was identified by immunoprecipitation, time course, and N-formyl-[35S]methionine labeling studies to be the product of the initiation of protein synthesis at an internal site(s) located within the 3'-proximal RNA sequences. Utilization of this internal initiation site(s) on poliovirus RNA was abolished when reticulocyte lysate was supplemented with poliovirus-infected HeLa cell extract. Authentic P1-1a was also synthesized in reticulocyte lysate, indicating that correct 5'-proximal initiation of translation occurs in that system. We conclude that the deficiency of a component(s) of the reticulocyte lysate necessary for 5'-proximal initiation of poliovirus protein synthesis resulted in the ability of ribosomes to initiate translation on internal sequences. This aberrant initiation could be corrected by factors present in the HeLa cell extract. Apparently, under certain conditions, ribosomes are capable of recognizing internal sequences as authentic initiation sites.     

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.     

Evidence for the presence of an inhibitor on ribosomes in mouse L cells infected with mengovirus.

After infection of mouse L cells with mengovirus, there is a rapid inhibition of protein synthesis, a concurrent disaggregation of polysomes, and an accumulation of 80S ribosomes. These 80S ribosomes could not be chased back into polysomes under an elongation block. The infected-cell 80S-ribosome fraction contained twice as much initiator methionyl-tRNA and mRNA as the analogous fraction from uninfected cells. Since the proportion of 80S ribosomes that were resistant to pronase digestion also increased after infection, these data suggest that the accumulated 80S ribosomes may be in the form of initiation complexes. The specific protein synthetic activity of polysomal ribosomes also decreased with time of infection. However, the transit times in mock-infected and infected cells remained the same. Cell-free translation systems from infected cells reflected the decreased protein synthetic activity of intact cells. The addition of reticulocyte initiation factors to such systems failed to relieve the inhibition. Fractionation of the infected-cell lysate revealed that the ribosomes were the predominant target affected. Washing the infected-cell ribosomes with 0.5 M KCI restored their translational activity. In turn, the salt wash from infected-cell ribosomes inhibited translation in lysates from mock-infected cells. The inhibitor in the ribosomal salt wash was temperature sensitive and micrococcal nuclease resistant. A model is proposed wherein virus infection activates (or induces the synthesis of) an inhibitor that binds to ribosomes and stops translation after the formation of the 80S-ribosome initiation complex but before elongation. The presence of such an inhibitor on ribosomes could prevent them from being remobilized into polysomes in the presence of an inhibitor of polypeptide elongation.     

The effect of Escherichia coli ribosomal protein S1 on the translational specificity of bacterial ribosomes

Ribosomes from Gram-negative bacteria such as Escherichia coli exhibit non-specific translation of bacterial mRNAs. That is, they are able to translate mRNAs from a variety of sources in a manner independent of the "strength" of the Shine-Dalgarno region, in contrast to ribosomes from many Gram-positive bacteria, such as Bacillus subtilis, which show specific translation in only being able to translate other Gram-positive mRNA, or mRNAs that have "strong" Shine-Dalgarno regions. There is an evolutionary correlation between the translational specificity and the absence of a protein analogous to E. coli ribosomal protein S1. The specificity observed with B. subtilis ribosomes is a function of their 30 S subunit which lacks S1; translation of Gram-negative mRNA can occur with heterologous ribosomes containing the 30 S subunit of E. coli ribosomes and the 50 S subunit of B. subtilis ribosomes. However, the addition of E. coli S1 alone to B. subtilis ribosome does not overcome their characteristic inability to translate mRNA from Gram-negative organisms. By contrast, the removal of S1 from E. coli ribosomes results in translational behavior similar to that shown by B. subtilis ribosomes in that the S1-depleted E. coli ribosomes can translate mRNA from Gram-positive sources in the absence of added S1, although addition of S1 stimulates further translation of such mRNAs by the E. coli ribosomes.     

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.     

Translation of poly(U) on ribosomes from Streptomyces aureofaciens

Slowly cooled cells of Streptomyces aureofaciens contained mainly tight-couple ribosomes. Maximum rate of polyphenylalanine synthesis on ribosomes of S. aureofaciens was observed at 40°C, while cultures grew optimally at 28°C. Ribosomes of S. aureofaciens differed from those of E. coli in the amount of poly(U) required for maximum synthetic activity. The polyphenylalanine-synthesizing activity of E. coli ribosomes was about 3-times higher than that of S. aureofaciens ribosomes. The addition of protein S1 of E. coli or the homologous protein from S. aureofaciens had no stimulatory effect on the translation of poly(U). In order to localize alteration(s) of S. aureofaciens ribosomes in the elongation step of polypeptide synthesis we developed an in vitro system derived from purified elongation factors and ribosomal subunits. The enzymatic binding of Phe-tRNA to ribosomes of S. aureofaciens was significantly lower than the binding to ribosomes of E. coli. This alteration was mainly connected with the function of S. aureofaciens 50 S subunits. These subunits were not deficient in their ability to associate with 30 S subunits or with protein SL5 which is homologous to L7/L12 of E. coli.     

Macromolecular synthesis in Streptomyces antibioticus: in vitro systems for aminoacylation and translation from young and old cells.

In vitro systems for the aminoacylation of transfer ribonucleic acid (tRNA) and for polypeptide synthesis have been constructed from young (12-h cultures, not producing actinomycin) and old (48-h cultures, producing actinomycin) cells of Streptomyces antibioticus. When Escherichia coli aminoacyl-tRNA synthetases were used to acylate S. antibioticus tRNA's, it was observed that, per absorbance unit of tRNA, the tRNA's from 48-h cells had a lower ability to accept the amino acids, leucine, serine, pheynlalanine, methionine, and valine than did the tRNA's from 12-h cells. Individual differences were observed between aminoacyl-tRNA synthetases from 12-h cells and those from 48-h cells with respect to the rate and extent of aminoacylation of E. coli tRNA with the five amino acids listed above. In vitro systems for the synthesis of polyphenylalanine have been constructed from 12- and 48-h cells. Ribsomes and soluble enzymes from 12-h cells are more efficient than those from 48-h cells in supporting polyphenylalanine synthesis, and, although the activity of both systems can be stimulated by the addition of E. coli tRNA, the higher level of incorporation observed in the unstimulated 12-h system (ribosomes and soluble enzymes) is maintained. Indeed, the difference in capacity for polyphenylalanine synthesis between in vitro systems from 12- and 48-h cells is greater when the systems are maximally stimulated by E. coli tRNA. Cross-mixing experiments reveal that enzymes from 48-h cells support a slightly higher level of polyphenylalanine synthesis than enzymes from 12-h cells with ribosomes from either cell type, and that the ribosomes are the primary agents responsible for the decreased efficiency of the in vito system from 48-h cells are compared with that from 12-h cells. To determine whether ribosome-associated factors were responsible for the relative inefficiency of the ribosomes from 48-h cells in translation, salt-washed ribosomes from 12- and 48-h cells were examined for their abilities to catalyze polyphenylalanine synthesis. Even after salt washing, ribosomes from 12-h cells were about five times higher in specific activity (counts per minute of polyphenylalanine synthesized per absorbance at 260 nm of ribosomes) than equivalent amounts of ribosomes from 48-h cells. Analysis of the proteins of salt-washed ribosomes of the two cell types by acrylamide gel electrophoresis suggests that the relative amounts of individual proteins present on ribosomes from 12-h cells are different from the amounts present on ribosomes from 48-h cells. These results are discussed in terms of the regulation of translation in S. antibioticus.     

The cytotoxic activity of Shigella toxin. Evidence for catalytic inactivation of the 60 S ribosomal subunit

The cytotoxic test system for Shigella shigae toxin was improved and used to study the stability of the toxin to various pH values, temperature, and chemicals. Inhibition of protein synthesis is the first demonstrable effect in cells treated with Shigella toxin. This inhibition appears to be at the level of peptide chain elongation. An inhibition effect on cell-free protein synthesis is exhibited by toxin pretreated first with trypsin and then with dithiothreitol and 8 M urea or 1% sodium dodecyl sulfate. Ribosomes treated with toxin or its A1 fragment had lost most of their ability to polymerize [14C]phenylalanine in a poly(U)-dependent cell-free system. Salt-washed ribosomes in simple buffered solutions were inactivated at a rate of at least 40 ribosomes/(min) (A1 fragment). Addition of antitoxin immediately stopped further inactivation, but it did not reactivate the inactivated ribosomes. 60 S ribosomal subunits from toxin-treated ribosomes had a marked reduction in ability to support polyphenylanine synthesis, whereas 40 S subunits from toxin-treated ribosomes retained their activity. Toxin-treated ribosomes retained their ability to incorporate [3H]puromycin into growing peptide chains, indicating that the peptide bond formation is not the function inhibited.     

Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs.

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.     

Calcium-Dependent Interaction of Calmodulin with Human 80S Ribosomes and Polyribosomes

Ribosomes are the protein factories of every living cell. The process of protein translation is highly complex and tightly regulated by a large number of diverse RNAs and proteins. Earlier studies indicate that Ca(2+) plays a role in protein translation. Calmodulin (CaM), a ubiquitous Ca(2+)-binding protein, regulates a large number of proteins participating in many signaling pathways. Several 40S and 60S ribosomal proteins have been identified to interact with CaM, and here, we report that CaM binds with high affinity to 80S ribosomes and polyribosomes in a Ca(2+)-dependent manner. No binding is observed in buffer with 6 mM Mg(2+) and 1 mM EGTA that chelates Ca(2+), suggesting high specificity of the CaM-ribosome interaction dependent on the Ca(2+) induced conformational change of CaM. The interactions between CaM and ribosomes are inhibited by synthetic peptides comprising putative CaM-binding sites in ribosomal proteins S2 and L14. Using a cell-free in vitro translation system, we further found that these synthetic peptides are potent inhibitors of protein synthesis. Our results identify an involvement of CaM in the translational activity of ribosomes.     

HIV-2 genomic RNA contains a novel type of IRES located downstream of its initiation codon

Eukaryotic translation initiation begins with assembly of a 48S ribosomal complex at the 5' cap structure or at an internal ribosomal entry segment (IRES). In both cases, ribosomal positioning at the AUG codon requires a 5' untranslated region upstream from the initiation site. Here, we report that translation of the genomic RNA of human immunodeficiency virus type 2 takes place by attachment of the 48S ribosomal preinitiation complex to the coding region, with no need for an upstream 5' untranslated RNA sequence. This unusual mechanism is mediated by an RNA sequence that has features of an IRES with the unique ability to recruit ribosomes upstream from its core domain. A combination of translation assays and structural studies reveal that sequences located 50 nucleotides downstream of the AUG codon are crucial for IRES activity.     

Translation of Synthetic and Endogenous Messenger Ribonucleic Acid In Vitro by Ribosomes and Polyribosomes from Clostridium pasteurianum

Ribosomes and polyribosomes from Clostridium pasteurianum were isolated and their activities were compared with those of ribosomes from Escherichia coli in protein synthesis in vitro. C. pasteurianum ribosomes exhibited a high level of activity due to endogenous messenger ribonucleic acid (RNA). For translation of polyuridylic acid [poly(U)], C. pasteurianum ribosomes required a higher concentration of Mg(2+) and a much higher level of poly(U) than did E. coli ribosomes. Phage f2 RNA added to the system with C. pasteurianum ribosomes gave no significant stimulation of protein synthesis in a homologous system or with E. coli initiation factors. The 30S and 50S subunits prepared from C. pasteurianum ribosomes reassociated less readily than subunits from E. coli. The ability of the C. pasteurianum subunits to reassociated was found to be dependent upon the presence of a reducing agent during preparation and during analysis of the reassociation products. In heterologous combinations, E. coli 30S subunits associated readily with C. pasteurianum 50S subunits to form 70S particles, but C. pasteurianum 30S subunits and E. coli 50S subunits did not associate. In poly(U) translation, E. coli 30S subunits were active in combination with 50S subunits from either E. coli or C. pasteurianum, but C. pasteurianum 30S subunits were not active in combination with either type of 50S subunits. Polyribosomes prepared from C. pasteurianum were very active in protein synthesis, and well-defined ribosomal aggregates as large as heptamers could be seen on sucrose gradients. An attempt was made to demonstrate synthesis in vitro of ferredoxin.     

A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits

Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.     

mRNA helicase activity of the ribosome

Most mRNAs contain secondary structure, yet their codons must be in single-stranded form to be translated. Until now, no helicase activity has been identified which could account for the ability of ribosomes to translate through downstream mRNA secondary structure. Using an oligonucleotide displacement assay, together with a stepwise in vitro translation system made up of purified components, we show that ribosomes are able to disrupt downstream helices, including a perfect 27 base pair helix of predicted T(m) = 70 degrees . Using helices of different lengths and registers, the helicase active site can be localized to the middle of the downstream tunnel, between the head and shoulder of the 30S subunit. Mutation of residues in proteins S3 and S4 that line the entry to the tunnel impairs helicase activity. We conclude that the ribosome itself is an mRNA helicase and that proteins S3 and S4 may play a role in its processivity.     

Dom34‐Hbs1 mediated dissociation of inactive 80S ribosomes promotes restart of translation after stress

Following translation termination, ribosomal subunits dissociate to become available for subsequent rounds of protein synthesis. In many translation‐inhibiting stress conditions, e.g. glucose starvation in yeast, free ribosomal subunits reassociate to form a large pool of non‐translating 80S ribosomes stabilized by the ‘clamping’ Stm1 factor. The subunits of these inactive ribosomes need to be mobilized for translation restart upon stress relief. The Dom34‐Hbs1 complex, together with the Rli1 NTPase (also known as ABCE1), have been shown to split ribosomes stuck on mRNAs in the context of RNA quality control mechanisms. Here, using in vitro and in vivo methods, we report a new role for the Dom34‐Hbs1 complex and Rli1 in dissociating inactive ribosomes, thereby facilitating translation restart in yeast recovering from glucose starvation stress. Interestingly, we found that this new role is not restricted to stress conditions, indicating that in growing yeast there is a dynamic pool of inactive ribosomes that needs to be split by Dom34‐Hbs1 and Rli1 to participate in protein synthesis. We propose that this provides a new level of translation regulation.     

Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli

Many contacts between the ribosome and its principal substrates, tRNA and mRNA, involve universally conserved rRNA nucleotides, implying their functional importance in translation. Here, we measure the in vivo translation activity conferred by substitution of each 16S rRNA base believed to contribute to the A or P site. We find that the 30S P site is generally more tolerant of mutation than the 30S A site. In the A site, A1493C or any substitution of G530 or A1492 results in complete loss of translation activity, while A1493U and A1493G decrease translation activity by >20-fold. Among the P-site nucleotides, A1339 is most critical; any mutation of A1339 confers a >18-fold decrease in translation activity. Regarding all other P-site bases, ribosomes harboring at least one substitution retain considerable activity, >10% that of control ribosomes. Moreover, several sets of multiple substitutions within the 30S P site fail to inactivate the ribosome. The robust nature of the 30S P site indicates that its interaction with the codon-anticodon helix is less stringent than that of the 30S A site. In addition, we show that G1338A suppresses phenotypes conferred by m(2)G966A and several multiple P-site substitutions, suggesting that adenine at position 1338 can stabilize tRNA interaction in the P site.     

ARC-1, a sequence element complementary to an internal 18S rRNA segment, enhances translation efficiency in plants when present in the leader or intercistronic region of mRNAs

The sequences of different plant viral leaders with known translation enhancer ability show partial complementarity to the central region of 18S rRNA. Such complementarity might serve as a means to attract 40S ribosomal subunits and explain in part the translation-enhancing property of these sequences. To verify this notion, we designed β-glucuronidase (GUS) mRNAs differing only in the nature of 10 nt inserts in the center of their 41 base leaders. These were complementary to consecutive domains of plant 18S rRNA. Sucrose gradient analysis revealed that leaders with inserts complementary to regions 1105–1114 and 1115–1124 (‘ARC-1’) of plant 18S rRNA bound most efficiently to the 40S ribosomal subunit after dissociation from 80S ribosomes under conditions of high ionic strength, a treatment known to remove translation initiation factors. Using wheat germ cell-free extracts, we could demonstrate that mRNAs with these leaders were translated more than three times more efficiently than a control lacking such a complementarity. Three linked copies of the insert enhanced translation of reporter mRNA to levels comparable with those directed by the natural translation enhancing leaders of tobacco mosaic virus and potato virus Y RNAs. Moreover, inserting the same leaders as intercistronic sequences in dicistronic mRNAs substantially increased translation of the second cistron, thereby revealing internal ribosome entry site activity. Thus, for plant systems, the complementary interaction between mRNA leader and the central region of 18S rRNA allows cap-independent binding of mRNA to the 43S pre-initiation complex without assistance of translation initiation factors.     

Candida albicans and three other Candida species contain an elongation factor structurally and functionally analogous to elongation factor 3.

A cell-free poly(U)-dependent translation elongation system from Candida albicans is ATP-dependent due to the presence of an elongation factor 3 (EF3)-like activity. Saccharomyces cerevisiae ribosomes added to a C. albicans postribosomal supernatant (PRS) supported poly(U)-dependent elongation, suggesting that the C. albicans lysate contained a soluble translation factor functionally analogous to the S. cerevisiae translation factor EF-3. The presence of EF-3 in C. albicans was confirmed by Western blotting using an antibody raised against S. cerevisiae EF-3. This antibody was also used to screen a selection of Candida species, all of which possessed EF-3 with molecular mass in the range of 110-130 kDa.