Formation Of Ribosomal Initiation Complex Research Articles

Escherichia coli ribosomal protein S1 unfolds structured mRNAs onto the ribosome for active translation initiation.

Author SummaryGene expression is regulated at multiple levels, including the decision of whether or not to translate a mRNA. This phenomenon, known as translational regulation, allows rapid changes in cellular concentrations of proteins and is well suited to the adjustment of cellular growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5′ untranslated regions that modulate the accessibility of the mRNA to the small ribosomal 30S subunit and so are directly involved in this regulatory process. Structured mRNAs must interact with the 30S subunit in a two-step pathway whereby the docking of a folded mRNA is followed by an accommodation step that involves unfolding of these structures. However, it is not known how the ribosome unfolds mRNA structures to promote translation initiation, nor which ribosomal factors are responsible for this activity. We demonstrate that the first three domains of ribosomal protein S1 endow the 30S subunit with an RNA chaperone activity that is essential for the binding and unfolding of structured mRNAs, allowing the correct positioning of the initiation codon for translation. However, ribosomal protein S1 is not required for all mRNAs and acts differently depending on the type of regulatory elements present in a given mRNA. In all, we have shown that ribosomal protein S1 provides an RNA-melting activity to the exit site of the 30S decoding channel and confers some plasticity on the ribosome to initiate translation of mRNAs.

Control of Translation and miRNA-Dependent Repression by a Novel Poly(A) Binding Protein, hnRNP-Q

Author SummaryThe regulation of mRNA translation and stability is of paramount importance for almost every cellular function. In eukaryotes, the poly(A) binding protein (PABP) is a central regulator of both global and mRNA-specific translation. PABP simultaneously interacts with the 3′ poly(A) tail of the mRNA and the eukaryotic translation initiation factor 4G (eIF4G). These interactions circularize the mRNA and stimulate translation. PABP also regulates specific mRNAs by promoting miRNA-dependent deadenylation and translational repression. A key step in understanding PABP's functions is to identify factors that affect its association with the poly(A) tail. Here we show that the cytoplasmic isoform of the mouse heterogeneous nuclear ribonucleoprotein Q (hnRNP-Q2/SYNCRIP), which exhibits binding preference to poly(A), interacts with the poly(A) tail by default when PABP binding is inhibited. In addition, hnRNP-Q2 competes with PABP for binding to the poly(A) tail. Depleting hnRNP-Q2 stimulates translation in cell-free extracts and in cultured cells, in agreement with its function as translational repressor. In addition, hnRNP-Q2 impeded miRNA-mediated deadenylation and repression of target mRNAs, which requires PABP. Thus, competition from hnRNP-Q2 provides a novel mechanism by which multiple functions of PABP are regulated. This regulation could play important roles in various biological processes, such as development, viral infection, and human disease.

Direct Monitoring of Initiation Factor Dynamics through Formation of 30S and 70S Translation-Initiation Complexes on a Quartz Crystal Microbalance

Translation initiation is a dynamic and complicated process requiring the building a 70S initiation complex (70S-IC) composed of a ribosome, mRNA, and an initiator tRNA. During the formation of the 70S-IC, initiation factors (IFs: IF1, IF2, and IF3) interact with a ribosome to form a 30S initiation complex (30S-IC) and a 70S-IC. Although some spectroscopic analyses have been performed, the mechanism of binding and dissociation of IFs remains unclear. Here, we employed a 27 MHz quartz crystal microbalance (QCM) to evaluate the process of bacterial IC formation in translation initiation by following frequency changes (mass changes). IFs (IF1, IF2, and IF3), N-terminally fused to biotin carboxyl carrier protein (bio-BCCP), were immobilized on a Neutravidin-covered QCM plate. By using bio-BCCP-IF2 immobilized to the QCM, three steps of the formation of ribosomal initiation complex could be sequentially observed as simple mass changes in real time: binding of a 30S complex to the immobilized IF2, a recruitment of 50S to the 30S-IC, and formation of the 70S-IC. The kinetic parameters implied that the release of IF2 from the 70S-IC could be the rate-limiting step in translation initiation. The IF3-immobilized QCM revealed that the affinity of IF3 for the 30S complex decreased upon the addition of mRNA and fMet-tRNA(fMet) but did not lead to complete dissociation from the 30S-IC. These results suggest that IF3 binds and stays bound to ICs, and its interaction mode is altered during the formation of 30S-IC and 70S-IC and is finally induced to dissociate from ICs by 50S binding. This methodology demonstrated here is applicable to investigate the role of IFs in translation initiation driven by other pathways.

General RNA-binding proteins have a function in poly(A)-binding protein-dependent translation

The interaction between the poly(A)-binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA-binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB-1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB-1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP-dependent after the addition of YB-1. In this system, eIF4E binding to the cap structure is inhibited by YB-1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB-1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB-1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP.

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.

Internal Initiation of Translation of Bovine Viral Diarrhea Virus RNA

Initiation of translation on the bovine viral diarrhea virus (BVDV) internal ribosomal entry site (IRES) was reconstituted in vitro from purified translation components to the stage of 48S ribosomal initiation complex formation. Ribosomal binding and positioning on this mRNA to form a 48S complex did not require the initiation factors eIF4A, eIF4B, or eIF4F, and translation of this mRNA was resistant to inhibition by a trans-dominant eIF4A mutant that inhibited cap-mediated initiation of translation. The BVDV IRES contains elements that are bound independently by ribosomal 40S subunits and by eukaryotic initiation factor (eIF) 3, as well as determinants that mediate direct attachment of 43S ribosomal complexes to the initiation codon.

Ribosome Initiation Complex Formation with the Pseudoknotted α Operon Messenger RNA

The Escherichia coli α mRNA has a complex pseudoknot secondary structure that forms the recognition site for a translational repressor, ribosomal protein S4, and also encompasses the regulated ribosome binding site. To find out whether the pseudoknot is a stable structure under the conditions of ribosome initiation complex formation, thermal denaturation of the RNA was monitored by calorimetry and ultraviolet light hyperchromicity. The secondary structure formed by the coding region melts in a single transition and has a stability of -7·4 kcal/mol at 37°C (5 mM-Mg2+, 100 mM-Na+, pH 7·0). A broad transition with tm ≈ 38°C may be a rearrangement of pseudoknot secondary or tertiary structure.Using reverse transcriptase primer extension assays ("toeprints") to measure the kinetics of ternary 30 S subunit-tRNAmetf -α mRNA translational initiation complex formation, we find a fast and a slow phase in the reaction. The fraction reacting rapidly is sensitive to temperature and mutations in the mRNA. We interpret these results in terms of "active" and "inactive" mRNA conformation that are trapped by 30 S subunits and react rapidly or slowly with tRNAmetf, respectively; the active form is predominant above 37°C. The binary 30 S-mRNA complex in the inactive form stops MMLV reverse transcriptase near the 3′ edge of the pseudoknot structure, apparently by stabilizing the pseudoknot.We propose the following mechanism for translational initiation with the α mRNA. The intact pseudoknot stimulates 30 S subunit binding, at low temperatures, but prevents proper binding of tRNAmetf. The inactive to active transition of the pseudoknot, which may be related to the 38°C transition seen in melting experiments, is required for tRNAmetf to pair with the anticodon and is rate-limiting for initiation complex formation at lower temperatures. A novel feature of this proposal is that mRNA structure affects a kinetic step in initiation complex formation, as well as ribosome binding affinity.

Early effects of dimethylnitrosamine on the initiation of protein synthesis in mouse liver.

1. Dimethylnitrosamine (37.5 mg/kg body wt.) was administered to mice by a single intraperitoneal injection, and the early effects on protein synthesis and related functions were studied in a liver S-30 system. 2. The incorporation of [14C]leucine into protein decreased rapidly after dimethylnitrosamine administration. The effect was associated with a decreased ability of the system to utilize methionyl-tRNAfMet and formyl-methionyl-tRNAfMet for 80 S ribosomal initiation-complex formation (primary initiation), and a loss of poly(A)-containing RNA from the postmicrosomal fraction. All the three effects developed simultaneously, and were clearly demonstrable within 15 min. 3. Initiation-complex formation in the polyribosomal fraction (re-initiation) was decreased to the same extent as the primary initiation, indicating that the initiation defect was not a result of the decrease in free mRNA. 4. The inhibition of initiation was only manifest at the joining of the 40 S pre-initiation complex to 60 S ribosomal subunits. It was not a result of methionyl-tRNAfMet deacylation. The functions between the formation of the methionyl-tRNAfMet-containing 80 S ribosomal complex and the first translocation on the ribosome were not involved, since the incorporation of formylmethionine into N-terminal polypeptides decreased to the same extent as the 80 S initiation-complex formation. 5. Inhibitors of protein synthesis (cycloheximide and pactamycin) decreased poly(A)-containing RNA in the postmicrosomal fraction in a similar way to dimethylnitrosamine.

Inhibition of 40S--Met--tRNAfMet ribosomal initiation complex formation by vaccinia virus.

Infection with vaccinia virus (a poxvirus) quickly and efficiently shuts off host protein synthesis in the presence of actinomycin D (refs 3--5) or cycloheximide. The cellular messenger RNA apparently remains stable in the infected cells exposed to inhibitors of viral gene transcription. In some cases vaccinia viral RNA or poly(A) synthesis have been implicated in the establishment of this effect. However, in the presence of cordycepin (3-deoxyadenosine) which blocks viral gene transcription and cytoplasmic poly(A) synthesis, cellular protein synthesis is still efficiently inhibited in vaccinia virus-infected cells. This shutoff is also observed in vitro, in the corresponding cell-free extracts, and in a reticulocyte lysate. Therefore the shutoff of host protein synthesis is probably mediated by a factor associated with vaccinia virions. We now report that the formation of the 40S--Met-tRNAfMet initiation complex is inhibited in cytoplasmic extracts derived from vaccinia virus-infected cells exposed to cordycepin to block viral gene expression. A similar inhibition is found in reticulocyte lysates incubated with purified vaccinia cores, confirming the hypothesis that the factor associated with the viral cores is responsible for the inhibition observed in vaccinia virus-infected cells exposed to inhibitors of transcription.

The effects of deacylated transfer ribonucleic acid on polypeptide-chain initiation in Ehrlich-ascites-tumour-cell extracts [proceedings

In intact Ehrlich ascites-tumour cells the rate of protein synthesis is decreased by over 6096 in the absence of a single essential amino acid (van Venrooij et al., 1972). This effect is due largely to an inhibition of polypeptide-chain initiation, and is associated with polyribosome disaggregation and a decrease in the number of Met-tRNAme-40 S subunit initiation complexes (Pain & Henshaw, 1975). Cell-free extracts prepared from control and lysinedeprived cells show some of the characteristics of the cells from which they were derived. Protein synthesis in starved-cell extracts is decreased by up to 75% relative to that in fed-cell extracts. Starved-cell extracts also have a diminished ability to form initiation complexes between 40s subunits and Met-tRNAme. This defect is abolished by addition of highly purified initiation factor eIF-2 (Pain et al., 1980; Clemens et al., 1980). It has been suggested (Vaughan & Hansen, 1973; Kyner el al., 1973) that the inhibition of polypeptide-chain initiation seen in cells deprived of amino acids may be due to increases in the concentrations of deacylated tRNA species. To test whether. increased concentrations of deacylated tRNA could directly inhibit polypeptide-chain initiation, the effect of adding periodate-oxidized tRNA (which cannot be charged with amino acids) to cell-free extracts from Ehrlich ascites-tumour cells was examined. Cell-free extracts from Ehrlich ascites-tumour cells were prepared, and analysis of labelled initiation complexes by sucrose-gradient centrifugation was performed as described by Pain et al. (1980). Deacylated calf liver tRNA was subjected to periodate oxidation as described by Vaughan & Hansen (1973). Addition of deacylated oxidized tRNA to cell-free extracts From Ehrlich ascites-tumour cells in the presence of 1 mMemetine to prevent polypeptide-chain elongation causes only a 10-2096 decrease in the concentration of Met-tRNAmet-40 S subunit initiabon complexes, at concentrations between 20 and 1OOpg of added tRNA/ml (Table 1). This small inhibition is not concentration-dependent. A much greater effect is seen on Met-tRNAme-80S ribosome initiation-complex formation. Addition of 20pg of deacylated tRNA/ml causes an 85% decrease in the concentration of Met-tRNAMet-80 S ribosome complexes. At higher concentrations of added tKNA Met-tRNAmet-80S ribosome complexes are completely abolished (Table 1). Addition of deacylated oxidized tRNA to cell-free extracts in the absence of emetine, where protein synthesis is occurring, causes an accumulation of Met-tRNAme-40S subunit initiation complexes. This implies impairment of a stage after MettRNAWCt-40S subunit initiation-complex formation, possibly 40s subunit-60s subunit joining. However, h this situation the concentrations of complexes seen just reflect the balance between their rate of formation and their rate of utilization, and the results are more difficult to interpret than those of experiments performed in the presence of an inhibitor of elongation. We have shown that concentrations of deacylated oxidized tRNA in the range 20-100pg/ml cause only a small decrease in the Concentration of Met-tRNAwd-40S subunit initiationcomplex formation. Since the concentration of Met-tRNAme40s subunit initiation complexes in extracts from lysinedeprived cells is decreased by up to 77% compared with that in extracts from control cells (Pain et al., 1980; Clemens et al., 1980), the small decline in initiation complexes seen with deacylated tRNA is not enough to account for the differences between fed-cell and starved-cell systems. It would seem therefore that an increase in the concentration of deacylated tRNA after amino acid deprivation is not the direct cause of the inhibition of this step of polypeptide-chain initiation in Ehrlich ascitestumour cells under these circumstances. Deacylated tRNA is strongly inhibitory to Met-tRNAmet-80s ribosome initiation-complex formation. This effect may be due to impairment of the conversion of Met-tRNAm-40S subunit initiation complexes into Met-tRNAmet-80S ribosome initiation complexes or to a destabilization of the latter complexes. The accumulation of Met-tRNAmet40S subunit initiation complexes seen in the presence of deacylated tRNA under protein-synthesis conditions is consistent with the former model. These results are also in agreement with the findings obtained by Kyner et al. (1973), who reported an inhibition of Met-tRNAmd-80S ribosome initiation-complex formation by uncharged tRNA in a fractionated cell-free system. As deacylated tRNA is unlikely to be directly responsible for the inhibition of polypeptide-chain initiation at the stage of Met-tRNAme40s subunit initiation-complex formation, the mechanism by which this part of the protein-synthesizing machinery of Ehrlich ascites-tumour cells recognizes the lack of one amino acid remains to be elucidated. This work is supported by grants from the Medical Research Council and the Cancer Research campaign. Clemens, M. J., Henshaw, E. C. & Pain, V. M. (1980) Blochem. Soc. Trans. 8,350-351

Inhibition of Qβ RNA 70S ribosome initiation complex formation by an oligonucleotide complementary to the 3′ terminal region of E. coli 16S ribosomal RNA

IT has been proposed that a key event in the recognition of a prokaryotic protein initiation site by the ribosome is the interaction between the 3′-terminal sequence of the 16S rRNA and a purine-rich sequence preceding the initiator triplet by three to nine nucleotides1,2 This hypothesis predicts that if the 3′-terminal region of the 16S rRNA were blocked, binding of the ribosome to the initiator region of an mRNA should be diminished or prevented, while binding to the trinucleotide A-U-G should not be affected We have shown that the oligonucleotide A-G-A-G-G-A-G-G-UOH (P-2a), eight bases of which are complementary to the sequence A-C-C-U-C-C-U-U-AOH at the 3′-termmal region of 16S rRNA (ΔG = −16.9 kcal mol−1 calculated as described in Table 1), binds tightly to the 30S ribosome and is released as a complex with the 3′-terminal 49-mer of 16S rRNA after digestion with cloacin DF13 (ref. 3). We report here that this oligonucleotide either strongly inhibits Qβ RNA binding to 70S ribosomes or decreases the stability of the initiation complex formed in its presence As we found that the formation of complexes of pA-U-G and 70S ribosomes was, not inhibited by the oligonucleotide, we conclude that inhibition (or destabilisation) of Qβ RNA binding is not due to an effect on the interaction between fMet-tRNA and the initiation triplet or on the association of the 30S and 50S ribosome subunits.