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.     

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.     

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.     

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.     

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.     

A kinetic light-scattering study of the binding of wheat germ protein synthesis initiation factor 3 to 40S ribosomal subunits and 80S ribosomes

The rate constants for eucaryotic initiation factor 3 (eIF3) association and dissociation with 40S ribosomal subunits and 80S monosomes have been determined. These rate constants were determined by laser light scattering with unmodified eIF3. The affinity of eIF3 for 40S subunits is about 30-fold greater than for 80S ribosomes. This difference in affinity resides mainly in the association rate constants. Rate constants of 8.8 X 10(7) and 7.3 X 10(6) M-1 s-1 were obtained for eIF3 binding to 40S subunits and 80S ribosomes, respectively. From thermodynamic cycles, the affinity of eIF3-40S subunits for 60S subunits is about 30-fold lower than free 40S subunits for 60S subunits. A calculation shows that under these conditions and assuming simple equilibria, approximately 12% of ribosomal subunits would associate via a reaction of 40S-eIF3 with 60S subunits as opposed to a path where eIF3 dissociates from the 40S subunits prior to association with 60S subunits.     

Interaction of translation initiation factor IF1 with the E. coli ribosomal A site

Initiation Factor 1 (IF1) is required for the initiation of translation in Escherichia coli. However, the precise function of IF1 remains unknown. Current evidence suggests that IF1 is an RNA-binding protein that sits in the A site of the decoding region of 16 S rRNA. IF1 binding to 30 S subunits changes the reactivity of nucleotides in the A site to chemical probes. The N1 position of A1408 is enhanced, while the N1 positions of A1492 and A1493 are protected from reactivity with dimethyl sulfate (DMS). The N1-N2 positions of G530 are also protected from reactivity with kethoxal. Quantitative footprinting experiments show that the dissociation constant for IF1 binding to the 30 S subunit is 0.9 microM and that IF1 also alters the reactivity of a subset of Class III sites that are protected by tRNA, 50 S subunits, or aminoglycoside antibiotics. IF1 enhances the reactivity of the N1 position of A1413, A908, and A909 to DMS and the N1-N2 positions of G1487 to kethoxal. To characterize this RNA-protein interaction, several ribosomal mutants in the decoding region RNA were created, and IF1 binding to wild-type and mutant 30 S subunits was monitored by chemical modification and primer extension with allele-specific primers. The mutations C1407U, A1408G, A1492G, or A1493G disrupt IF1 binding to 30 S subunits, whereas the mutations G530A, U1406A, U1406G, G1491U, U1495A, U1495C, or U1495G had little effect on IF1 binding. Disruption of IF1 binding correlates with the deleterious phenotypic effects of certain mutations. IF1 binding to the A site of the 30 S subunit may modulate subunit association and the fidelity of tRNA selection in the P site through conformational changes in the 16 S rRNA.     

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.     

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.     

The PRC-barrel domain of the ribosome maturation protein RimM mediates binding to ribosomal protein S19 in the 30S ribosomal subunits

The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes. A DeltarimM mutant is defective in 30S maturation and accumulates 17S rRNA. To study the interaction of RimM with the 30S and its involvement in 30S maturation, RimM amino acid substitution mutants were constructed. A mutant RimM (RimM-YY-->AA), containing alanine substitutions for two adjacent tyrosines within the PRC beta-barrel domain, showed a reduced binding to 30S and an accumulation of 17S rRNA compared to wild-type RimM. The (RimM-YY-->AA) and DeltarimM mutants had significantly lower amounts of polysomes and also reduced levels of 30S relative to 50S compared to a wild-type strain. A mutation in rpsS, which encodes r-protein S19, suppressed the polysome- and 16S rRNA processing deficiencies of the RimM-YY-->AA but not that of the DeltarimM mutant. A mutation in rpsM, which encodes r-protein S13, suppressed the polysome deficiency of both rimM mutants. Suppressor mutations, found in either helices 31 or 33b of 16S rRNA, improved growth of both the RimM-YY-->AA and DeltarimM mutants. However, they suppressed the 16S rRNA processing deficiency of the RimM-YY-->AA mutant more efficiently than that of the DeltarimM mutant. Helices 31 and 33b are known to interact with S13 and S19, respectively, and S13 is known to interact with S19. A GST-RimM but not a GST-RimM(YY-->AA) protein bound strongly to S19 in 30S. Thus, RimM likely facilitates maturation of the region of the head of 30S that contains S13 and S19 as well as helices 31 and 33b.     

The protein composition of reconstituted 30S ribosomal subunits: the effects of single protein omission.

Using reverse phase HPLC, we have been able to quantify the protein compositions of reconstituted 30S ribosomal subunits, formed either with the full complement of 30S proteins in the reconstitution mix or with a single protein omitted. We denote particles formed in the latter case as SPORE (single protein omission reconstitution) particles. An important goal in 30S reconstitution studies is the formation of reconstituted subunits having uniform protein composition, preferably corresponding to one copy of each protein per reconstituted particle. Here we describe procedures involving variation of the protein:rRNA ratio that approach this goal. In SPORE particles the omission of one protein often results in the partial loss in uptake of other proteins. We also describe procedures to increase the uptake of such proteins into SPORE particles, thus enhancing the utility of the SPORE approach in defining the role of specific proteins in 30S structure and function. The losses of proteins other than the omitted protein provide a measure of protein:protein interaction within the 30S subunit. Most of these losses are predictable on the basis of other such measures. However, we do find evidence for several long-range protein:protein interactions (S6:S3, S6:S12, S10:S16, and S6:S4) that have not been described previously.     

Effect of spermidine on N-formylmethionyl-tRNA binding to 30S ribosomal subunits and on N-formylmethionyl-tRNA dependent polypeptide synthesis.

The spermidine stimulation of AUG dependent F-met-tRNA binding to 30S ribosomal subunits and polypeptide synthesis was greater than that of GUG dependent F-met-tRNA binding and polypeptide synthesis. Spermidine stimulation of polypeptide synthesis was greatest when AUG(U)_n was used as a template.     

Cross-links between ribosomal proteins of 30S subunits in 70S tight couples and in 30S subunits

Ribosome 70S tight couples and 30S subunits derived from them were modified with 2-iminothiolane under conditions where about two sulfhydryl groups per protein were added to the ribosomal particles. The 70S and 30S particles were not treated with elevated concentrations of NH4Cl, in contrast to those used in earlier studies. The modified particles were oxidized to promote disulfide bond formation. Proteins were extracted from the cross-linked particles by using conditions to preclude disulfide interchange. Disulfide-linked protein complexes were fractionated on the basis of charge by electrophoresis in polyacrylamide/urea gels at pH 5.5. The proteins from sequential slices of the urea gels were analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Final identification of proteins in cross-linked complexes was made by radioiodination of the proteins, followed by two-dimensional polyacrylamide/urea gel electrophoresis. Attention was focused on cross-links between 30S proteins. We report the identification of 27 cross-linked dimers and 2 trimers of 30S proteins, all but one of which were found in both 70S ribosomes and free 30S subunits in similar yield. Seven of the cross-links, S3-S13, S13-S21, S14-S19, S7-S12, S9-S13, S11-S21, and S6-S18-S21, have not been reported previously when 2-iminothiolane was used. Cross-links S3-S13, S13-S21, S7-S12, S11-S21, and S6-S18-S21 are reported for the first time. The identification of the seven new cross-links is illustrated and discussed in detail. Ten of the dimers reported in the earlier studies of Sommer & Traut (1976) [Sommer, A., & Traut, R. R. (1976) J. Mol. Biol. 106, 995-1015], using 30S subunits treated with high salt concentrations, were not found in the experiments reported here.     

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.