Binding of protein synthesis initiation factor IF1 to 30 S ribosomal subunits: effects of other initiation factors and identification of proteins near the binding site.

The effects of other components of the initiation complex on Escherichia coli initiation factor IFI binding to 30 S ribosomal subunits were studied. Binding of [¹⁴C]IF1 in the absence of other initiation complex components was slight. Addition of either IF2 or IF3 stimulated binding to a variable extent. Maximum binding was observed when both IF2 and IF3 were present. Addition of GTP, fMet-tRNA, and phage R17 RNA caused little or no further stimulation of [¹⁴C]IF1 binding. A maximum of 0.5 molecule of [¹⁴C]IF1 bound per 30 S subunit in the presence of an excess of each of the three factors over 30 S subunits.Complexes of 30 S subunits, [¹⁴C]IF1, IF2, and IF3 were treated with the bifunctional protein cross-linking reagent dimethyl suberimidate in order to identify the ribosomal proteins near the binding site for IF1. Non-cross-linked [¹⁴C]IF1 was removed from the complexes by sedimentation through buffer containing a high salt concentration, and total protein was extracted from the pelleted particles. Approximately 12% of the [¹⁴C]IF1 was recovered in the pellet fraction. The mixture of cross-linked products was analyzed by polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Autoradiography of the gel showed radioactive bands with molecular weights of 21,000, 25,000, and many greater than 120,000. The results indicate that [¹⁴C]IF1 was cross-linked directly to at least two ribosomal proteins. Analysis of the cross-linked mixture by radioimmunodiffusion with specific antisera prepared against each of the 30 S ribosomal proteins showed radioactivity in the precipitin bands formed with antisera against S12 and S19, and in lower yield with those against S1 and S13. Antiserum against IF2 also showed [¹⁴C]IF1 in the precipitin band. The results show that [¹⁴C]IF1 was present in covalently cross-linked complexes containing 30 S ribosomal proteins S1, S12, S13 and S19, and initiation factor IF2. The same ribosomal proteins have been implicated in the binding sites for IF2 and IF3. The results suggest that the three initiation factors bind to the 30 S subunit at the same or overlapping sites.     

Cross-linking of initiation factor IF3 to proteins of the Escherichia coli 30 S ribosomal subunit

Complexes of 30 S subunits and [¹⁴C]IF3 were allowed to react with the protein cross-linking reagents, N,N′-p-phenylenedimaleimide or dimethylsuberimidate. Non-cross-linked IF3 was removed from the complex by centrifugation in a buffer containing a high salt concentration, and the total protein was extracted from the pelleted particles. The mixture of cross-linked products was analyzed by radioimmunodiffusion with antisera prepared against all of the individual 30 S ribosomal proteins. Radioactivity was found in the precipitin bands formed with antisera against ribosomal proteins S1, S11, S12, S13, S19 and S21. The results show that IF3 was present in covalent cross-linked complexes containing those 30 S ribosomal proteins and imply that they comprise or are near the binding site for initiation factor IF3.     

Photochemical cross-linking of translation initiation factor 3 to Escherichia coli 50S ribosomal subunits

Translation initiation factor 3 (IF-3) was bound noncovalently to Escherichia coli 50S ribosomal subunits. Irradiation of such complexes with near-ultraviolet light (greater than 285 nm) resulted in covalent attachment of initiation factor 3 to the 50S subunit. Photo-cross-linking attained its maximum level of 40% of that which was noncovalently bound after 90 min of irradiation. Cross-linking was abolished in the presence of either 0.5 M NH4C1 or 0.25 mM aurintricarboxylic acid, indicating that specific binding of initiation factor 3 to the ribosome was a prerequisite for subsequent covalent attachment. Further analysis showed that all the IF-3 was covalently bound to a small number of 50S subunit proteins. The major cross-linked proteins were identified as L2, L7/L12, L11, and L27 by immunochemical techniques. These results are discussed in light of the proposed mechanism for IF-3 function.     

Segmental flexibility in Escherichia coli ribosomal protein S1 as studied by fluorescence polarization.

Ribosomal protein S1 covalently reacts with approximately one equivalent of iodoacetylethylenediamine (1,5-napthol sulfonate (IAEDANS) or iodoacetylaminofluorescein (IAAF). The product AEDANS-S1 can bind to 30S ribosomal subunits lacking S1 as shown by polyacrylamide-agarose gel electrophoresis AEDANS-S1 and AAF-S1 when added back to S1-depleted 30S subunits modulate poly(U)-dependent polyphenylalanine synthesis in the presence of IF3 in a very similar way to unmodified S1. AEDANS-S1 also stimulates RI7-dependent fMet-tRNA binding to 1.0M NH4C1 washed ribosomes whereas AAF-S1 does not. Both static and nanosecond fluorescence polarization techniques were used to study the rotational motions of AEDANS-S1. Several previous studies had indicated that S1 is a highly extended protein which can be modeled by a prolate ellipsoid with an axial ratio of 10 to 1. However, the rotational correlation time we find is about half that expected for such a particle. This suggests that S1 is a flexible protein with at least two domains that can rotate independently.     

Terminal oxidation-reduction of N-acetyl-phenylalanyl-tRNA blocks initiation complex formation with Escherichia coli 30S ribosomal subunits

Terminally oxidized-reduced tRNA^Phe of yeast, exclusively acylated at the 2′-hydroxyl of the 3′-terminal ribose of the tRNA, is a useful model for investigating the stereospecificity of AA-tRNA in protein synthesis. In this work, the ability of N-acetyl-Phe-tRNA^ox-red to form an initiation complex with Escherichiacoli 30S ribosomal subunits was investigated. Thirty per cent of added control N-acetyl-Phe-tRNA was bound in a reaction dependent on initiation factors, GTP, and poly U, but no binding of the oxidized-reduced analog could be detected. These results imply that initiation complex formation may be specific for the 3′-ester of initiator tRNA.     

Escherichia coli initiation factor 3 protein binding to 30S ribosomal subunits alters the accessibility of nucleotides within the conserved central region of 16S rRNA

Translational initiation factor 3 (IF3) is an RNA helix destabilizing protein which interacts with strongly conserved sequences in 16S rRNA, one at the 3' terminus and one in the central domain. It was therefore of interest to identify particular residues whose exposure changes upon IF3 binding. Chemical and enzymatic probing of central domain nucleotides of 16S rRNA in 30S ribosomal subunits was carried out in the presence and absence of IF3. Bases were probed with dimethyl sulfate (DMS), at A(N-1), C(N-3), and G(N-7), and with N-cyclohexyl-N'-[2-(N-methyl-4-morpholinio)ethyl] carbodiimide p-toluenesulfonate (CMCT), at G(N-1) and U(N-3). RNase T1 and nuclease S1 were used to probe unpaired nucleotides, and RNase V1 was used to monitor base-paired or stacked nucleotides. 30S subunits in physiological buffers were probed in the presence and absence of IF3. The sites of cleavage and modification were detected by primer extension. IF3 binding to 30S subunits was found to reduce the chemical reactivity and enzymatic accessibility of some sites and to enhance attack at other sites in the conserved central domain of 16S rRNA, residues 690-850. IF3 decreased CMCT attack at U701 and U793 and V1 attack at G722, G737, and C764; IF3 enhanced DMS attack at A814 and V1 attack at U697, G833, G847, and G849. Many of these central domain sites are strongly conserved and with the conserved 3'-terminal site define a binding domain for IF3 which correlates with a predicted cleft in two independent models of the 30S ribosomal subunit.     

Effect of Escherichia coli initiation factors on the kinetics of N-Acphe-tRNAPhe binding to 30S ribosomal subunits. A fluorescence stopped-flow study

The mechanism of binding of N-AcPhe-tRNAPhe (yeast) to poly(U)-programmed Escherichia coli 30S ribosomal subunits and the effect of individual initiation factors (IF-1, IF-2, and IF-3) and GTP on this process have been studied by fluorescence stopped-flow kinetic measurements. The formation of the ternary complex was followed by an increase of both intensity and polarization of the fluorescence of a proflavin label located in the anticodon loop of the tRNA. The effect of the initiation factors and GTP is to increase the velocity of ternary complex formation (about 400-fold at 7 mM Mg2+). In the presence of the three initiation factors and GTP the formation of the ternary complex could be resolved into two partial reactions: a fast apparently second-order step (k12 = 5 x 10(6) M-1 s-1, k21 = 1.4 s-1) followed by a slow rearrangement step (k23 less than or equal to 0.1 s-1). The data suggest a mechanism in which the ternary complex is formed by at least two rearrangements of an initially formed preternary complex. The accelerating effects of both IF-2 and IF-3 can be understood by assuming a synergistic allosteric action of the factors on the 30S ribosomal subunit, whereas IF-1 appears to act indirectly by influencing the other two factors.     

Interaction of ribosomal protein S1 and initiation factor IF3 with the 3' major domain and the decoding site of the 30S subunit of Escherichia coli.

We have studied the effect of the binding of ribosomal protein S1 and initiation factor IF3 on the accessibility of nucleotide residues 584-1506 in the small subunit of the Escherichia coli ribosome. Protein S1 strongly decreases RNase V1 attack at G1164, in hairpin 40 of the 3' major domain, and weakly decreases DMS attack at C1302, in the central loop of the 3' major domain, and at A1503, in the 3' minor domain. It also weakly increases the DMS reactivity of A1004, in the 3' major domain, and of A901, in the central domain. Factor IF3 strongly decreases RNase V1 attack (but not dimethyl sulfate attack) at A1408, in the decoding site, and weakly protects A1500, in the 3' minor domain and near the colicin E3 cleavage site. Neomycin does not interfere with this effect of IF3, but IF3 interferes with the protective effect of neomycin against dimethyl sulfate attack at A1408.     

Binding of initiation factor 2 and initiator tRNA to the Escherichia coli 30S ribosomal subunit induces allosteric transitions in 16S rRNA.

The specific effect of the binding of IF2 and initiator fMET-tRNA(fMet) on Escherichia coli 16S rRNA has been probed by phosphate alkylation with ethylnitrosourea. The results show that IF2 does not significantly shield portions of 16S rRNA but induces both reductions and enhancements of reactivity scattered in the entire molecule. Most of them are topographically constrained in a region corresponding to the cleft, the lateral protrusion, and the part of the head facing the protrusion (positions 694, 771, 791, 1225, 1268, 1398, 1401, 1504, and 1527). Weak effects are also observed in distant parts of the subunit (positions 301, 302, 492, and 1428). All the reactivity changes induced by the binding of IF2 are still observed in the presence of the initiator tRNA and AUG as messenger. The additional changes induced by the tRNA are mostly centered around the cleft-head-lateral protrusion region, near positions affected by IF2 binding. Most of the changes correspond to reduced reactivities (positions 791, 1222, 1263, 1393, 1395, 1430, 1431, 1504, 1528, and 1529), while enhanced reactivities are observed at positions 708, 709, and 1398. Functional implications are discussed, which stress the dynamic properties of the ribosome.     

High resolution 1H-n.m.r. study of the interaction between initiation factor IF1 and 30S ribosomal subunits

Interaction between Escherichia coli translational initiation factor IF1 (mol. wt. 8119) and 30S ribosomal subunits was followed by high resolution 1H-n.m.r. spectroscopy. Upon gradual addition of increasing yet largely sub-stoichiometric amounts of biologically active deuterated 30S ribosomal subunits, selective line broadenings and chemical shift changes were observed against the background of the gradual disappearance of the whole spectrum. At the highest 30S:IF1 ratio attained (0.25), all the resonance lines were broadened beyond meaningful detection. This behaviour, which can be partly reversed by increasing the ionic strength and/or the temperature, is due to the interaction between IF1 and the 30S ribosomal subunits, and can be explained by the existence of a medium-fast exchange dynamics between free and bound factor. The selective effects observed during titration with 30S ribosomal subunits shed some light on the mode of interaction of IF1 with 30S ribosomal subunits. At least one of the two His residues of the factor appears to be involved in the binding, since it undergoes a low-field change of chemical shift and becomes totally immobilized in the IF1-30S complex. Also strongly implicated in the interaction with 30S are more than one Ser and Arg residue and probably one lysine. Additional effects of the interaction of IF1 with ribosomes are a drastic reduction in the intensity of the ring current upfield shifted methyl resonances and mobilization of a previously rotationally hindered phenylalanine ring.     

Position of protein S1 in the 30 S ribosomal subunit of Escherichia coli

The results of neutron distance measurement involving ribosomal protein S1 from Escherichia coli are reported. These data provide a position for S1 on the small ribosomal subunit. They also indicate that S1, bound to the ribosome, has a radius of gyration of 60 to 65 Å, suggesting that its axial ratio in the bound state is similar to that it has as a free molecule in solution; namely, 10: 1. The implications of these results for our understanding of the mode of action of S1 are discussed.     

The interaction between initiation factor 3 and 30 S ribosomal subunits studied by high-resolution 1H NMR spectroscopy

The interaction between Escherichia coli translational initiation factor 3 (IF-3) (Mr = 20668) and 30 S ribosomal subunits or fragmented 16 S rRNA was followed by 1H NMR spectroscopy. Upon addition of increasing yet largely substoichiometric amounts of deuterated 30 S ribosomal subunits, selective line broadenings and some chemical shift changes were observed. These effects can be fully reversed by increasing the temperature and/or the ionic strength. The selective line broadenings, which are explained by a medium-fast to fast exchange dynamics between free and bound IF-3 with loss of internal mobility of the protons, shed light on the amino acid residues of IF-3 involved in or affected by the binding to the 30 S subunits. Some effects (i.e. implication of 1 tyrosine, 1 phenylalanine, and some arginine and lysine residues) are seen with both 30 S subunits and rRNA while others (i.e. implication of a second tyrosine or phenylalanine residue of a group of hydrophobic residues and, possibly, of the single histidine residue), seen only or preferentially with 30 S subunits, may reflect additional interactions exclusively occurring at the ribosomal level.     

Binding of eukaryotic initiation factor 3 to ribosomal 40S subunits and its role in ribosomal dissociation and anti-association

The multisubunit eukaryotic initiation factor (eIF) 3 plays various roles in translation initiation that all involve interaction with 40S ribosomal subunits. eIF3 can be purified in two forms: with or without the loosely associated eIF3j subunit (eIF3j+ and eIF3j-, respectively). Although unlike eIF3j+, eIF3j- does not bind 40S subunits stably enough to withstand sucrose density gradient centrifugation, we found that in addition to the known stabilization of the eIF3/40S subunit interaction by the eIF2*GTP*Met-tRNA(i)Met ternary complex, eIF3j-/40S subunit complexes were also stabilized by single-stranded RNA or DNA cofactors that were at least 25 nt long and could be flanked by stable hairpins. Of all homopolymers, oligo(rU), oligo(dT), and oligo(dC) stimulated the eIF3/40S subunit interaction, whereas oligo(rA), oligo(rG), oligo(rC), oligo(dA), and oligo(dG) did not. Oligo(U) or oligo(dT) sequences interspersed by other bases also promoted this interaction. The ability of oligonucleotides to stimulate eIF3/40S subunit association correlated with their ability to bind to the 40S subunit, most likely to its mRNA-binding cleft. Although eIF3j+ could bind directly to 40S subunits, neither eIF3j- nor eIF3j+ alone was able to dissociate 80S ribosomes or protect 40S and 60S subunits from reassociation. Significantly, the dissociation/anti-association activities of both forms of eIF3 became apparent in the presence of either eIF2-ternary complexes or any oligonucleotide cofactor that promoted eIF3/40S subunit interaction. Ribosomal dissociation and anti-association activities of eIF3 were strongly enhanced by eIF1. The potential biological role of stimulation of eIF3/40S subunit interaction by an RNA cofactor in the absence of eIF2-ternary complex is discussed.     

Immune electron microscopic localization of dinitrophenyl-modified ribosomal protein S19 in reconstituted Escherichia coli 30 S subunits using antibodies to dinitrophenol

Escherichia coli small ribosomal subunits have been reconstituted from RNA and high performance liquid chromatography-purified proteins including protein S19 that had been modified at its amino-terminal proline residue with 1-fluoro-2,4-dinitrobenzene. As detailed in the accompanying paper (Olah, T. V., Olson, H. M., Glitz, D. G., and Cooperman, B. S. (1988) J. Biol. Chem. 263, 4795-4800), dinitrophenyl (DNP)-S19 was efficiently incorporated into the site ordinarily occupied by S19. Antibodies to DNP bound effectively to the reconstituted subunits and did not cause dissociation of the modified protein from the subunit. Electron microscopy of the immune complexes was used to localize the modified protein on the subunit surface. More than 95% of the antibody binding sites seen were consistent with a single location of protein S19 on the upper portion or head of the subunit, on the surface that faces the 50 S particle in a 70 S ribosome, and in an area relatively distant from the subunit platform. The S19 site is close to the region in which 30 S subunits are photoaffinity labeled with puromycin. Protein S19 is thus near protein S14 in the small subunit and in proximity to the peptidyl transferase center of the 70 S ribosome.     

Purification and characterization of protein synthesis initiation factors IF1, IF2, and IF3 from Escherichia coli.

A simple procedure is described for the purification in high yields of protein synthesis initiation factors IF1, IF2, and IF3 from Escherichia coli strain MRE 600. IF2 was separated from IF1 and IF3 by ammonium sulfate fractionation and was purified by column chromatography on phosphocellulose and diethylaminoethyl (DEAE) Sephadex. IF1 and IF3 were separated by phosphocellulose column chromatography. IF1 was purified by molecular sieve chromatography, and IF3 by phosphocellulose column chromatography in urea buffer. Each factor was analyzed by sodium dodecyl sulfate or urea polyacrylamide gel electrophoresis and was greater than 98% pure. Only one form of IF1 and IF3 was found, with molecular weights of 8,500 and 22,500, respectively. Two forms of IF2 were isolated: IF2a with a molecular weight of 118,000 and IF2b with a molecular weight of 90,000. The amino acid composition of each factor was determined, and their stimulation in a variety of assays for initiation of protein synthesis is reported.