The cryo-EM structure of a translation initiation complex from Escherichia coli

The 70S ribosome and its complement of factors required for initiation of translation in E. coli were purified separately and reassembled in vitro with GDPNP, producing a stable initiation complex (IC) stalled after 70S assembly. We have obtained a cryo-EM reconstruction of the IC showing IF2*GDPNP at the intersubunit cleft of the 70S ribosome. IF2*GDPNP contacts the 30S and 50S subunits as well as fMet-tRNA(fMet). IF2 here adopts a conformation radically different from that seen in the recent crystal structure of IF2. The C-terminal domain of IF2 binds to the single-stranded portion of fMet-tRNA(fMet), thereby forcing the tRNA into a novel orientation at the P site. The GTP binding domain of IF2 binds to the GTPase-associated center of the 50S subunit in a manner similar to EF-G and EF-Tu. Additionally, we present evidence for the localization of IF1, IF3, one C-terminal domain of L7/L12, and the N-terminal domain of IF2 in the initiation complex.     

Suppression of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era,an essential GTPase in Escherichia coli

Era is a small GTP-binding protein and essential for cell growth in Escherichia coli. It consists of two domains: N-terminal GTP-binding and C-terminal RNA-binding KH domains. It has been shown to bind to 16S rRNAs and 30S ribosomal subunits in vitro. Here, we report that a precursor of 16S rRNA accumulates in Era-depleted cells. The accumulation of the precursors is also seen in a cold-sensitive mutant, E200K, in which the mutation site is located in the C-terminal domain. The major precursor molecule accumulated seems to be 17S rRNA, containing extra sequences at both 5' and 3' ends of 16S rRNA. Moreover, the amounts of both 30S and 50S ribosomal subunits relative to the amount of 70S monosomes increase in Era-depleted and E200K mutant cells. The C-terminal KH domain has a high structural similarity to the RbfA protein, a cold shock protein that also specifically associates with 30S ribosomal subunits. RbfA is essential for cell growth at low temperature, and a precursor of 16S rRNA accumulates in an rbfA deletion strain. The 16S rRNA precursor seems to be identical in size to that accumulated in Era mutant cells. Surprisingly, the cold-sensitive cell growth of the rbfA deletion cells was partially suppressed by overproduction of the wild-type Era. The C-terminal domain alone was not able to suppress the cold-sensitive phenotype, whereas Era-dE, which has a 10-residue deletion in a putative effector region of the N-terminal domain, functioned as a more efficient suppressor than the wild-type Era. It was found that Era-dE suppressed defective 16S rRNA maturation, resuming a normal polysome profile to reduce highly accumulated free 30S and 50S subunits in the rbfA deletion cells. These results indicate that Era is involved in 16S rRNA maturation and ribosome assembly.     

Binding of human ribosomal protein p40 and its truncated mutants to the small ribosomal subunit

Ribosomal protein SA (rpSA), or p40, is a structural element of the small subunit of the eukaryotic ribosome. The N-terminal and central parts of rpSA are homologous to prokaryotic S2, whereas its C-terminal part is specific to eukaryotes. Preparations of 40S ribosomal subunits isolated from full-term human placenta proved to be deficient in SA to a varying extent. To study the rpSA binding to human 40S subunits, recombinant rpSA and its mutant forms with N-and C-terminal deletions were synthesized. The full-size and N-truncated rpSA variants bound to 40S subunits, while deletion of the C-terminal domain completely abolished the binding.     

Identification of the ribosome binding sites of translation initiation factor IF3 by multidimensional heteronuclear NMR spectroscopy.

Titrations of Escherichia coli translation initiation factor IF3, isotopically labeled with 15N, with 30S ribosomal subunits were followed by NMR by recording two-dimensional (15N,1H)-HSQC spectra. In the titrations, intensity changes are observed for cross peaks belonging to amides of individual amino acids. At low concentrations of ribosomal subunits, only resonances belonging to amino acids of the C-domain of IF3 are affected, whereas all those attributed to the N-domain are still visible. Upon addition of a larger amount of 30S subunits cross peaks belonging to residues of the N-terminal domain of the protein are also selectively affected. Our results demonstrate that the two domains of IF3 are functionally independent, each interacting with a different affinity with the ribosomal subunits, thus allowing the identification of the individual residues of the two domains involved in this interaction. Overall, the C-domain interacts with the 30S subunits primarily through some of its loops and alpha-helices and the residues involved in ribosome binding are distributed rather symmetrically over a fairly large surface of the domain, while the N-domain interacts mainly via a small number of residues distributed asymmetrically in this domain. The spatial organization of the active sites of IF3, emerging through the comparison of the present data with the previous chemical modification and mutagenesis data, is discussed in light of the ribosomal localization of IF3 and of the mechanism of action of this factor.     

The interaction of mammalian mitochondrial translational initiation factor 3 with ribosomes: evolution of terminal extensions in IF3mt

Mammalian mitochondrial initiation factor 3 (IF3_mt) has a central region with homology to bacterial IF3. This homology region is preceded by an N-terminal extension and followed by a C-terminal extension. The role of these extensions on the binding of IF3_mt to mitochondrial small ribosomal subunits (28S) was studied using derivatives in which the extensions had been deleted. The K_d for the binding of IF3_mt to 28S subunits is ~30 nM. Removal of either the N- or C-terminal extension has almost no effect on this value. IF3_mt has very weak interactions with the large subunit of the mitochondrial ribosome (39S) (K_d = 1.5 μM). However, deletion of the extensions results in derivatives with significant affinity for 39S subunits (K_d = 0.12−0.25 μM). IF3_mt does not bind 55S monosomes, while the deletion derivative binds slightly to these particles. IF3_mt is very effective in dissociating 55S ribosomes. Removal of the N-terminal extension has little effect on this activity. However, removal of the C-terminal extension leads to a complex dissociation pattern due to the high affinity of this derivative for 39S subunits. These data suggest that the extensions have evolved to ensure the proper dissociation of IF3_mt from the 28S subunits upon 39S subunit joining.     

Initiation factor IF 2 binds to the alpha-sarcin loop and helix 89 of Escherichia coli 23S ribosomal RNA

During initiation of protein synthesis in bacteria, translation initiation factor IF2 is responsible for the recognition of the initiator tRNA (fMet-tRNA). To perform this function, IF2 binds to the ribosome interacting with both 30S and 50S ribosomal subunits. Here we report the topographical localization of translation initiation factor IF2 on the 70S ribosome determined by base-specific chemical probing. Our results indicate that IF2 specifically protects from chemical modification two sites in domain V of 23S rRNA, namely A2476 and A2478, and residues around position 2660 in domain VI, the so-called sarcin-ricin loop. These footprints are generated by IF2 regardless of the presence of fMet-tRNA, GTP, mRNA, and IF1. IF2 causes no specific protection of 16S rRNA. We observe a decreased reactivity of residues A1418 and A1483, which is an indication that the initiation factor has a tightening effect on the association of ribosomal subunits. This result, confirmed by sucrose density gradient analysis, seems to be a universally conserved property of IF2.     

Translation initiation factor IF2 interacts with the 30 S ribosomal subunit via two separate binding sites

The functional properties of the two natural forms of Escherichia coli translation initiation factor IF2 (IF2alpha and IF2beta) and of an N-terminal deletion mutant of the factor (IF2DeltaN) lacking the first 294 residues, corresponding to the entire N-terminal domain, were analysed comparatively. The results revealed that IF2alpha and IF2beta display almost indistinguishable properties, whereas IF2DeltaN, although fully active in all steps of the translation initiation pathway, displays functional activities having properties and requirements distinctly different from those of the intact molecule. Indeed, binding of IF2DeltaN to the 30 S subunit, IF2DeltaN-dependent stimulation of fMet-tRNA binding to the ribosome and of initiation dipeptide formation strongly depend upon the presence of IF1 and GTP, unlike with IF2alpha and IF2beta. The present results indicate that, using two separate active sites, IF2 establishes two interactions with the 30 S ribosomal subunit which have different properties and functions. The first site, located in the N domain of IF2, is responsible for a high-affinity interaction which "anchors" the factor to the subunit while the second site, mainly located in the beta-barrel module homologous to domain II of EF-G and EF-Tu, is responsible for the functional ("core") interaction of IF2 leading to the decoding of fMet-tRNA in the 30 S subunit P-site. The first interaction is functionally dispensable, sensitive to ionic-strength variations and essentially insensitive to the nature of the guanosine nucleotide ligand and to the presence of IF1, unlike the second interaction which strongly depends upon the presence of IF1 and GTP.     

The major ribosome binding site of Escherichia coli ribosomal protein S1 is located in its N-terminal segment

We have cleaved protein S1, which is the largest and the most elongated protein of the Escherichia coli ribosome, using cyanogen bromide and isolated two fragments that retain the functional domains of the intact molecule. The fragments (denoted S1-F2a and S1-F2b) showed molecular weights of 24,000 and 22,500 by dodecyl sulphate/polyacrylamide gel electrophoresis. Fragment F2a is shown to be the N-terminal segment containing about 32% of the peptide chain length of S1. Fragment F2b is derived from another (probably C-terminal) region of S1.Fragment F2a binds to 30 S ribosomal subunits with a strength and specificity comparable to the binding of intact S1. It also binds to matrix-bound poly(U) but the binding is salt-sensitive, unlike the binding of intact S1. Fragment F2b binds only very weakly to poly(U) and does not bind to 30 S subunits. These results are discussed with respect to the ribosome binding domain(s) of protein S1 and the possible interdependence of the multiple functional domains in this large protein.     

The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain

Efficient protein synthesis in bacteria requires initiation factor 2 (IF2), elongation factors Tu (EF-Tu) and G (EF-G), and release factor 3 (RF3), each of which catalyzes a major step of translation in a GTP-dependent fashion. Previous reports have suggested that recruitment of factors to the ribosome and subsequent GTP hydrolysis involve the dimeric protein L12, which forms a flexible "stalk" on the ribosome. Using heteronuclear NMR spectroscopy we demonstrate that L12 binds directly to the factors IF2, EF-Tu, EF-G, and RF3 from Escherichia coli, and map the region of L12 involved in these interactions. Factor-dependent chemical shift changes show that all four factors bind to the same region of the C-terminal domain of L12. This region includes three strictly conserved residues, K70, L80, and E82, and a set of highly conserved residues, including V66, A67, V68 and G79. Upon factor binding, all NMR signals from the C-terminal domain become broadened beyond detection, while those from the N-terminal domain are virtually unaffected, implying that the C-terminal domain binds to the factor, while the N-terminal domain dimer retains its rotational freedom mediated by the flexible hinge between the two domains. Factor-dependent variations in linewidths further reveal that L12 binds to each factor with a dissociation constant in the millimolar range in solution. These results indicate that the L12-factor complexes will be highly populated on the ribosome, because of the high local concentration of ribosome-bound factor with respect to L12.     

Structure-function analysis of Escherichia coli translation initiation factor IF3: tyrosine 107 and lysine 110 are required for ribosome binding.

Translation initiation factor IF3 is required for peptide chain initiation in Escherichia coli. IF3 binds directly to 30S ribosomal subunits ensuring a constant supply of free 30S subunits for initiation complex formation, participates in the kinetic selection of the correct initiator region of mRNA, and destabilizes initiation complexes containing noninitiator tRNAs. The roles that tyrosine 107 and lysine 110 play in IF3 function were examined by site-directed mutagenesis. Tyrosine 107 was changed to either phenylalanine (Y107F) or leucine (Y107L), and lysine 110 was converted to either arginine (K110R) or leucine (K110L). These single amino acid changes resulted in a reduced affinity of IF3 for 30S subunits. Association equilibrium constants (M-1) for 30S subunit binding were as follows: wild-type, 7.8 x 10(7); Y107F, 4.1 x 10(7); Y107L, 1 x 10(7); K110R, 5.1 x 10(6); K110L, < 1 x 10(2). The mutant IF3s were similarly impaired in their abilities to specifically select initiation complexes containing tRNA(fMet). Toeprint analysis indicated that 5-fold more Y107L or K110R protein was required for proper initiator tRNA selection. K110L protein was unable to mediate this selection even at concentrations up to 10-fold higher than wild type. The results indicate that tyrosine 107 and lysine 110 are critical components of the ribosome binding domain of IF3 and, furthermore, that dissociation of complexes containing noninitiator tRNAs requires prior binding of IF3 to the ribosomes.     

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.     

X-ray crystallography shows that translational initiation factor IF3 consists of two compact alpha/beta domains linked by an alpha-helix.

The structures of the two domains of translational initiation factor IF3 from Bacillus stearothermophilus have been solved by X-ray crystallography using single wavelength anomalous scattering and multiwavelength anomalous diffraction. Each of the two domains has an alpha/beta topology, with an exposed beta-sheet that is reminiscent of several ribosomal and other RNA binding proteins. An alpha-helix that protrudes out from the body of the N-terminal domain towards the C-terminal domain suggests that IF3 consists of two RNA binding domains connected by an alpha-helix and that it may bridge two regions of the ribosome. This represents the first high resolution structural information on a translational initiation factor.     

Preparation and characterization of two monoclonal antibodies against different epitopes in Escherichia coli ribosomal protein L7/L12

Two monoclonal antibodies with specificities for Escherichia coli 50 S ribosomal subunit protein L7/L12 were isolated. The antibodies and Fab fragments thereof were purified by affinity chromatography using solid-phase coupled L7/L12 protein as the immunoadsorbent. The two antibodies were shown to recognize different epitopes; one in the N-terminal and the other in the C-terminal domain of protein L7/L12. Both intact antibodies strongly inhibited polyuridylic acid-directed polyphenylalanine synthesis, ribosome-dependent GTPase activity, and the binding of elongation factor EF-G to the ribosome. Ratios of antibody to ribosome of 4:1 or less were effective in inhibiting these activities. Neither antibody prevented the association of ribosomal subunits to form 70 S ribosomes. The Fab fragments showed similar effects.     

Binding of Escherichia coli protein synthesis initiation factor IF1 to 30S ribosomal subunits measured by fluorescence polarization

The interaction of initiation factor IF1 with 30S ribosomal subunits was measured quantitatively by fluorescence polarization. Purified IF1 was treated with 2-iminothiolane and N-[[(iodoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonic acid in order to prepare a covalent fluorescent derivative without eliminating positive charges on the protein required for biochemical activity. The fluorescent-labeled IF1 binds to 30S subunits and promotes the formation of N-formylmethionyl-tRNA complexes with 70S ribosomes. Analyses of mixtures of fluorescent-labeled IF1 and 30S ribosomal subunits with an SLM 4800 spectrofluorometer showed little change in fluorescence spectra or lifetimes upon binding, but a difference in polarization between free and bound forms is measurable. Bound to free ratios were calculated from polarization data and used in Scatchard plots to determine equilibrium binding constants and number of binding sites per ribosomal subunit. Competition between derivatized and nonderivatized forms of IF1 was quantified, and association constants for the native factor were determined: (5 +/- 1) X 10(5) M-1 with IF1 alone; (3.6 +/- 0.4) X 10(7) M-1 with IF3; (1.1 +/- 0.2) X 10(8) M-1 with IF2; (2.5 +/- 0.5) X 10(8) M-1 with both IF2 and IF3. In all cases, 0.9-1.1 binding sites per 30S subunit were detected. Divalent cations have little effect on affinities, whereas increasing monovalent cations inhibit binding. On the basis of the association constants, we predict that greater than 90% of native 30S subunits are complexed with all three initiation factors in intact bacterial cells.     

Solution structure of the C1-subdomain of Bacillus stearothermophilus translation initiation factor IF2

IF2 is one of three bacterial translation initiation factors that are conserved through all kingdoms of life. It binds the 30S and 50S ribosomal subunits, as well as fMet-tRNAf(Met). After these interactions, fMet-tRNAf(Met) is oriented to the ribosomal P-site where the first amino acid of the nascent polypeptide, formylmethionine, is presented. The C-terminal domain of Bacillus stearothermophilus IF2, which is responsible for recognition and binding of fMet-tRNAf(Met), contains two structured modules. Previously, the solution structure of the most C-terminal module, IF2-C2, has been elucidated by NMR spectroscopy and direct interactions between this subdomain and fMet-tRNAf(Met) were reported. In the present NMR study we have obtained the spectral assignment of the other module of the C-terminal domain (IF2-C1) and determined its solution structure and backbone dynamics. The IF2-C1 core forms a flattened fold consisting of a central four-stranded parallel beta-sheet flanked by three alpha-helices. Although its overall organization resembles that of subdomain III of the archaeal IF2-homolog eIF5B whose crystal structure had previously been reported, some differences of potential functional significance are evident.     

Structural Insights into ribosome recycling factor interactions with the 70S ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.     

The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control

The Escherichia coli autoregulatory ribosomal protein L20 consists of two structurally distinct domains. The C-terminal domain is globular and sits on the surface of the large ribosomal subunit whereas the N-terminal domain has an extended shape and penetrates deep into the RNA-rich core of the subunit. Many other ribosomal proteins have analogous internal or terminal extensions. However, the biological functions of these extended domains remain obscure. Here we show that the N-terminal tail of L20 is important for ribosome assembly in vivo. Indeed, a truncated version of L20 without its N-terminal tail is unable to complement the deletion of rplT, the gene encoding L20. In addition, this L20 truncation confers a lethal-dominant phenotype, suggesting that the N-terminal domain is essential for cell growth because it could be required for ribosome assembly. Supporting this hypothesis, partial deletions of the N-terminal tail of the protein are shown to cause a slow-growth phenotype due to altered ribosome assembly in vivo as large amounts of intermediate 40S ribosomal particles accumulate. In addition to being a ribosomal protein, L20 also acts as an autogenous repressor. Using L20 truncations, we also show that the N-terminal tail of L20 is dispensable for autogenous control.     

Ribosomal Interaction of Bacillus stearothermophilus Translation Initiation Factor IF2: Characterization of the Active Sites

InfB-encoded translation initiation factor IF2 contains a non-conserved N-terminal domain and two conserved domains (G and C) constituted by three (G1, G2 and G3) and two (C1 and C2) sub-domains. Here, we show that: (i) Bacillus stearothermophilus IF2 complements in vivo an Escherichia coli infB null mutation and (ii) the N-domain of B. stearothermophilus IF2, like that of E. coli IF2, provides a strong yet dispensable interaction with 30 S and 50 S subunits in spite of the lack of any size, sequence or structural homology between the N-domains of the two factors. Furthermore, the nature of the B. stearothermophilus IF2 sites involved in establishing the functional interactions with the ribosome was investigated by generating deletion, random and site-directed mutations within sub-domains G2 or G3 of a molecule carrying an H301Y substitution in switch II of the G2 module, which impairs the ribosome-dependent GTPase activity of IF2. By selecting suppressors of the dominant-lethal phenotype caused by the H301Y substitution, three independent mutants impaired in ribosome binding were identified; namely, S387P (in G2) and G420E and E424K (in G3). The functional properties of these mutants and those of the deletion mutants are compatible with the premise that IF2 interacts with 30 S and 50 S subunits via G3 and G2 modules, respectively. However, beyond this generalization, because the mutation in G2 resulted in a functional alteration of G3 and vice versa, our results indicate the existence of extensive “cross-talking” between these two modules, highlighting a harmonic conformational cooperation between G2 and G3 required for a functional interaction between IF2 and the two ribosomal subunits. It is noteworthy that the E424K mutant, which completely lacks GTPase activity, displays IF2 wild-type capacity in supporting initiation of dipeptide formation.     

Roles of the N- and C-terminal domains of mammalian mitochondrial initiation factor 3 in protein biosynthesis

Bacterial initiation factor 3 (IF3) is organized into N- and C-domains separated by a linker. Mitochondrial IF3 (IF3_mt) has a similar domain organization, although both domains have extensions not found in the bacterial factors. Constructs of the N- and C-domains of IF3_mt with and without the connecting linker were prepared. The K_d values for the binding of full-length IF3_mt and its C-domain with and without the linker to mitochondrial 28S subunits are 30, 60, and 95 nM, respectively, indicating that much of the ribosome binding interactions are mediated by the C-domain. However, the N-domain binds to 28S subunits with only a 10-fold lower affinity than full-length IF3_mt. This observation indicates that the N-domain of IF3_mt has significant contacts with the protein-rich small subunit of mammalian mitochondrial ribosomes. The linker also plays a role in modulating the interactions between the 28S subunit and the factor; it is not just a physical connector between the two domains. The presence of the two domains and the linker may optimize the overall affinity of IF3_mt for the ribosome. These results are in sharp contrast to observations with Escherichia coli IF3. Removal of the N-domain drastically reduces the activity of IF3_mt in the dissociation of mitochondrial 55S ribosomes, although the C-domain itself retains some activity. This residual activity depends significantly on the linker region. The N-domain alone has no effect on the dissociation of ribosomes. Full-length IF3_mt reduces the binding of fMet-tRNA to the 28S subunit in the absence of mRNA. Both the C-terminal extension and the linker are required for this effect. IF3_mt promotes the formation of a binary complex between IF2_mt and fMet-tRNA that may play an important role in mitochondrial protein synthesis. Both domains play a role promoting the formation of this complex.