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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

Mutations in 16S rRNA that suppress cold-sensitive initiation factor 1 affect ribosomal subunit association

A mutation in the infA gene encoding initiation factor 1 (IF1) gives rise to a cold-sensitive phenotype. An Escherichia coli strain with this mutation was used as a tool to select for second-site suppressors that compensate for the cold sensitivity and map specifically to rRNA. Several suppressor mutants with altered 16S rRNA that partially restore growth of an IF1 mutant strain in the cold were isolated and characterized. Suppressor mutations were found in helix (h)18, h32, h34 and h41 in 16S rRNA. These mutations are not clustered to any particular region in 16S rRNA and none overlap previously reported sites of interaction with IF1. While the isolated suppressors are structurally diverse, they are functionally related because all affect ribosomal subunit association in vivo. Furthermore, in vitro subunit-association experiments indicate that most of the suppressor mutations directly influence ribosomal subunit association even though none of these are confined to any of the known intersubunit bridges. These results are consistent with the model that IF1 is an rRNA chaperone that induces large-scale conformational changes in the small ribosomal subunit, and as a consequence modulates initiation of translation by affecting subunit association.     

Functional heterogeneity of the 30S ribosomal subunit of Escherichia coli. II. Effect of S21 on initiation

Summary Native 30S ribosomal subunits fromEscherichia coli are deficient in fractional protein S21, which is present on the monosome and polysome-derived 30S subunits. The presence of S21 prevents the binding of Fmet-tRNA if and only if 50S subunits are present. In contrast, proteins S2, S3 and S14 stimulate the binding of Fmet-tRNA. These results have been used to rationalize other data concerning the mechanism of Fmet-tRNA binding by ribosomes. In addition, the present data indicate that the 30S ribosomal subunits are heterogeneousin vivo as well asin vitro.Dedicated to Professor H. Veldstra on the occasion of his retirement from the chair of biochemistry of the University of Leiden.     

Interaction of translation initiation factor 3 with the 30S ribosomal subunit.

Hydroxyl radical footprinting and directed probing from Fe(II)-derivatized IF3 have been used to map the interaction of IF3 relative to 16S rRNA and tRNA(Met)(f) in the 30S ribosomal subunit. Our results place the two domains of IF3 on opposite sides of the initiator tRNA, with the C domain at the platform interface and the N domain at the E site. The C domain coincides with the location of helix 69 of 23S rRNA, explaining the ability of IF3 to block subunit association. The N domain neighbors proteins S7 and S11 and may interfere with E site tRNA binding. Our model suggests that IF3 influences initiator tRNA selection indirectly.     

The conformation of 16S RNA in the 30S ribosomal subunit from Escherichia coli.

The digestion of E. coli 16S RNA with a single-strand-specific nuclease produced two fractions separable by gel filtration. One fraction was small oligonucleotides, the other, comprising 67.5% of the total RNA, was highly structured double helical fragments of mol. wt. 7,600. There are thus about 44 helical loops of average size corresponding to 12 base pairs in each 16S RNA. 10% of the RNA could be digested from native 30S subunits. Nuclease attack was primarily in the intraloop single-stranded region but two major sites of attack were located in the interloop single-stranded regions. Nuclease digestion of unfolded subunits produced three classes of fragments, two of which, comprising 80% of the total RNA, were identical to fragments from 16S RNA. The third, consisting of 20% RNA, together with an equal weight of peotein, was a resistant core (sedimentation coefficient 7S).     

Cross-linking of streptomycin to the 16S ribosomal RNA of Escherichia coli

[3H]Dihydrostreptomycin was cross-linked to the 30S ribosomal subunit from Escherichia coli with the bifunctional reagent nitrogen mustard. The cross-linking primarily involved the 16S RNA. To localize the site of cross-linking of streptomycin to the 16S RNA, we hybridized RNA labeled with streptomycin to restriction fragments of the 16S RNA gene. Labeled RNA hybridized to DNA fragments corresponding to bases 892-917 and bases 1394-1415. These two segments of the ribosomal RNA must be juxtaposed in the ribosome, since there is a single binding site for streptomycin. This region has been implicated both in the decoding site and in the binding of initiation factor IF-3, indicating its functional importance.     

The Escherichia coli 30S ribosomal subunit; an optimized three-dimensional fit between the ribosomal proteins and the 16S RNA.

We have generated a computerized fit between the 3-dimensional map of the E.coli 30S ribosomal proteins, as determined by neutron scattering, and the recently published 3-dimensional model for the 16S RNA. To achieve this, the framework of coordinates for RNA-protein cross-link sites on the phosphate backbone in the RNA model was related to the corresponding framework of coordinates for the mass centres of the proteins by a least squares fitting procedure. The resulting structure, displayed on a computer graphics system, gives the first complete picture of the E.coli 30S ribosomal subunit showing both the proteins and the double-helical regions of the RNA. The root mean square distance between cross-link sites and protein centres is 32 A. The position of the mass centre of the combined double-helical regions was calculated from the model and compared with the position of the mass centre of the complete set of proteins. The two centres are displaced relative to one another by 20 A in the model structure, in good agreement with the experimental value of 25 A found by neutron scattering.     

Crosslinking studies on the organization of the 16 S ribosomal RNA within the 30 S Escherichia coli ribosomal subunit.

The location and frequency of RNA crosslinks induced by photoreaction of hydroxymethyltrimethylpsoralen with 30 S Escherichia coli ribosomal subunits have been determined by electron microscopy. At least seven distinct crosslinks between regions distant in the 16 S rRNA primary structure are seen in the inactive conformation of the 30 S particle. All correspond to crosslinked features seen when the free 16 S rRNA is treated with hydroxymethyltrimethylpsoralen. The most frequently observed crosslink occurs between residues near one end of the molecule and residues about 600 nucleotides away to generate a loop of 570 bases. The size and orientation of this feature indicate it corresponds to the crosslinked feature located at the 3′ end of free 16 S rRNA.When active 30 S particles are crosslinked in 5 mm-Mg²⁺, six of the seven features seen in the inactive 30 S particle can still be detected. However, the frequency of several of the features, and particularly the 570-base loop feature, is dramatically decreased. This suggests that the long-range contacts that lead to these crosslinks are either absent or inaccessible in the active conformation. Crosslinking results in some loss of functional activities of the 30 S particle. This is consistent with the notion that the presence of the crosslink that generates the 570-base loop traps the subunit in an inactive form, which cannot associate with 50 S particles.The arrangement of the interacting regions crosslinked by hydroxymethyltrimethylpsoralen suggests that the RNA may be organized into three general domains. A striking feature of the Crosslinking pattern is that three of the seven products involve regions near the 3′ end of the 16 S rRNA. These serve to tie together large sections of rRNA. Thus structural changes at the 3′ end could, in principle, be felt through the entire 30 S particle.