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.     

Circular dichroism analysis of the ribosomal binding of initiation factor IF-3

The circular dichroism spectra of Escherichia coli 30 S ribosomal subunits have been determined between 200 and 320 nm in the presence and in the absence of initiation factor IF-3. The addition of IF-3 did not produce any major alteration of the circular dichroism spectrum of the 30 S subunits between 320 and 240 nm, but resulted in an increase of the negative ellipticity between 240 and 205 nm. The effect was maximal for an IF-3:30 S molar ratio of approximately one, and further addition of IF-3 did not lead to a further increase of ellipticity. A similar effect was not seen when the 30 S ribosomal subunits were previously heat-inactivated to destroy their IF-3 binding capacity. These data indicate that the ribosomal binding of IF-3 may be accompanied by an increase in the secondary structure of the ribosomal proteins, but does not involve any major net change in the secondary structure of the rRNA.     

Nature of the ribosomal binding site for initiation factor 3 (IF-3)

$\text{In vitro}$ labelled IF-3 binds to both 16S and 23S rRNA but while one molecule of IF-3 binds to each 30S particle, binding to 50S particles is negligible. If proteins are removed by LiCl or CsCl treatment from either ribosomal subunit, however, binding specificity is lost and new “binding sites” appear on both ribosomal particles. Controlled RNase digestion of the 30S subunits does not cause the loss of any r-protein while controlled trypsin digestion results in the loss or degradation of several r-proteins; compared to the Phe-tRNA binding site, the binding site of IF-3 seems to be more sensitive to RNase than to trypsin digestion. Antibodies against single 30S r-proteins, which inhibit other ribosomal functions, do not prevent the binding of IF-3. RNA-binding dyes (acridine orange and pyronine) inhibit the binding of IF-3 to 30S ribosomal subunits. It is proposed that a segment of the 16S rRNA provides the binding site for IF-3 and that r-proteins confer specificity, restricting the number of available “binding sites”, and stabilize the 30S-IF-3 interaction.     

Euglena gracilis chloroplast initiation factor 2. Identification and initial characterization

The chloroplast protein synthesis factor responsible for the binding of fMet-tRNAMeti to chloroplast 30 S ribosomal subunits (IF-2chl) has been identified in whole cell extracts of Euglena gracilis. The IF-2chl activity is present in considerably higher amounts in extracts of light-grown cells than in extracts of dark-grown cells. About 90% of this activity is found in the postribosomal supernatant of the cell. Chromatography on phosphocellulose results in the partial purification of IF-2chl and separates the chloroplast factor from the cytoplasmic factor eIF-2A. The binding of fMet-tRNAMeti to chloroplast 30 S subunits is message-dependent as observed for prokaryotic systems. In addition, GTP stimulates the IF-2chl-dependent reaction 3-fold. The binding reaction shows broad monovalent and divalent cation optima. The activity of IF-2chl is stimulated 2-fold by the addition of either Escherichia coli IF-1 or IF-3, and 4-fold by the inclusion of both factors. Chloroplast IF-2 is quite active on the homologous 30 S ribosomal subunits but shows little activity on E. coli 30 S or wheat germ 40 S subunits.     

Activity of Euglena gracilis chloroplast ribosomes with procaryotic and eucaryotic initiation factors

A method that permits the preparation of Euglena gracilis chloroplast 30 S ribosomal subunits that are largely free of endogenous initiation factors and that are active in the binding of fMet-tRNA in response to poly(A, U, G), has been developed. These 30 S subunits have been tested for activity in initiation complex formation with initiation factors from both procaryotes and eucaryotes. We have observed that Escherichia coli IF-2 binds fMet-tRNA nearly as well to Euglena chloroplast ribosomal subunits as it does to its homologous subunits. Neither wheat germ eIF-2 nor Euglena eIF-2A can bind fMet-tRNA efficiently to Euglena chloroplast or E. coli 30 S subunits although both are active with wheat germ 40 S ribosomal subunits. Euglena chloroplast 68 S ribosomes will also bind the initiator tRNA. Both E. coli IF-2 and E. coli IF-3 stimulate this reaction on chloroplast ribosomes with approximately the same efficiency as they do on their homologous ribosomes. E. coli IF-1 enhances the binding of fMet-tRNA to the chloroplast 68 S ribosomes when either IF-2 or IF-3 is limiting. The chloroplast ribosomes unlike E. coli ribosomes show considerable activity over a broad range of Mg2+ ion concentrations.     

Resistance of bacterial protein synthesis to double-stranded RNA

Double-stranded RNA fails to inhibit the formation of translation initiation complexes on R17 bacteriophage RNA, overall synthesis of R17 proteins, or the ability of bacterial initiation factor IF-3 to prevent the association of 30S and 50S ribosomal subunits into single ribosomes. Yet, IF-3 can form complexes with double-stranded RNA. However, IF-3 binds to double-stranded RNA with lower apparent affinity than to either R17 RNA or 30S ribosomal subunits; this may explain the resistance of bacterial protein synthesis to double-stranded RNA.     

Interaction of Escherichia coli translation-initiation factor IF-1 with ribosomes

The interaction between Escherichia coli translation-initiation factor IF-1 and ribosomes was studied in binding experiments by Airfuge centrifugation. IF-1 binds to the 30S, but not to the 50S, ribosomal subunit and its binding is strongly stimulated by IF-3 and IF-2, either alone or in combination. From the dependence of the Kd of the 30S-subunit--IF-1 complex on ionic strength, it can be concluded that IF-1 binds primarily via an ionic interaction, most likely with the 16S rRNA, with the minimum number of ion pairs involved being 2.7-3.6. The 30S-subunit--IF-1 interaction is unaffected by temperature changes between 11 degrees C and 44 degrees C and is thus accompanied by a negligible enthalpy change. It is concluded that the interaction is an entropy-driven process triggered mainly by the release of counter ions from the RNA phosphates. Titration of 30S-subunit--IF-1 complexes with 50S subunits causes the ejection of the factor indicating that IF-1 is released from the ribosomes during the subunit association step which marks the transition from a 30S-initiation-complex to a 70S initiation complex.     

Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16S ribosomal RNA of Escherichia coli. IV. The effect of the methylgroups on ribosomal subunit interaction.

The effect of the presence or absence of the methylgroups of the m2(6)Am2(6)A sequence near the 3' end of 16S rRNA of Escherichia coli on the interaction of the ribosomal subunits has been studied, using wild-type (methylated) and mutant (unmethylated) ribosomes. Subunit exchange experiments and competitive association experiments show a strong preference of the 50S subunit for association with methylated 30S subunits. The results indicate that the equilibrium constant of the reaction 70S in equilibrium with 30S + 50S is dependent on the methylgroups; mutant 30S.50S couples are less stable than wild-type 30S.50S couples. It is postulated that the methylgroups also stimulate the interaction between 30S subunits and initiation factor IF-3.     

Isolation of ribosomal subunits containing intact rRNA from human placenta: estimation of functional activity of 80S ribosomes.

We have elaborated a method for the isolation of ribosomal subunits from fresh unfrozen human placenta containing intact rRNA and a complete set of ribosomal proteins. Activity of 80S ribosomes obtained by reassociation of 40S and 60S subunits in nonenzymatic poly(U)-dependent binding of Phe-tRNA(Phe) was equal to 80% (above 1.5 mol [14C]Phe-tRNA(Phe) is coupled to 1 mol of ribosomes). The activity of 80S ribosomes in poly(U)-directed synthesis of polyphenylalanine was tested in a polysome-free protein-synthesizing system from rabbit reticulocytes. About 100 mol of phenylalanine residue was polymerized by a mole of ribosomes at a rate of 0.83 residues per minute in this system (2 h, 37 degrees C).     

Participation of initiation factor IF-3 in the binding of AcPhe-tRNA to the 30S ribosomal subunit

Initiation factor IF-3 is required in addition to IF-1 and IF-2 for maximal initial rate of poly(U)-directed binding of AcPhe-tRNA to 30S ribosomal subunits of $\text{E. coli}$. Incubation periods longer than 10 sec, by which time the reaction is virtually over, progressively obscure the requirement for IF-3 in AcPhe-tRNA binding. IF-3 also stimulates the poly(A, G, U)-directed binding of fMet-tRNA to the 30S ribosomal subunit, but in this case, significant stimulation can still be observed even with extended incubation. These results indicate that IF-3 functions similarly in the translation of synthetic mRNA, as it does with natural mRNA, participating in ribosome dissociation and in the formation of the initiation complex from the 30S ribosomal subunit.     

Effect of polypeptide initiation factors on the spermidine stimulation of initiation complex formation

The AUG- and MS2 RNA-dependent fMet-tRNA binding to 30S ribosomal subunits was stimulated by spermidine with any individual or combination of initiation factors capable of participating in the formation of an initiation complex. When 70S ribosomes were used instead of 30S ribosomal subunits, IF-3 was necessary for spermidine stimulation of the complex formation.     

Structure-function relationship in Escherichia coli initiation factors. Biochemical and biophysical characterization of the interaction between IF-2 and guanosine nucleotides

Equilibrium dialysis and protection from heat inactivation and proteolysis show that initiation factor 2 (IF-2) interacts not only with GTP but also with GDP and that its conformation is changed upon binding of either nucleotide. The apparent Ka (at 25 degrees C) for the IF-2 X GDP and IF-2 X GTP complexes was 8.0 X 10(4) and 7.0 X 10(3) M(-1), respectively. The lower affinity for GTP is associated with a more negative delta S0. The interaction, monitored by 1HNMR spectroscopy, is characterized by fast exchange and results in line broadening and downfield shift of the purine C-8 and ribose C-1' protons of GTP as well as of the beta, gamma-methylene protons of (beta-gamma-methylene)guanosine 5'-triphosphate. The interaction of guanosine nucleotides with IF-2 requires an H bond donor (or acceptor) group at position C-2 of the purine and involves the beta- and/or gamma-phosphate of the nucleotide while the ribose 2'-OH group or the integrity of the furan ring are less critical. IF-2 binds to ribosomal particles with decreasing affinity: 30 S greater than 70 S greater than 50 S. GTP and GDP have no effect on the binding to 70 S. GTP stimulates the binding to the 30 S and depresses somewhat the binding to the 50 S subunits; GDP has the opposite effect. These results seem to rule out that the release of IF 2 from 70 S is due to a "GDP-conformation" of the factor incompatible with its permanence on the ribosome. The rate and the extent of 30 S initiation complex formation are approximately 2-fold higher with IF-2 X GTP than with IF-2 alone. At low concentrations of IF-2 and 30 S subunits, GDP inhibits this reaction, acting as a strong competitive inhibitor of GTP (Ki = 1.25 X 10(-5)m) and preventing IF-2 from binding to the ribosomal subunit.     

Chain initiation factor 3 crosslinks to E. coli 30S and 50S ribosomal subunits and alters the UV absorbance spectrum of 70S ribosomes

We report a direct procedure to determine the proteins near the IF-3 binding site in purified 30S and 50S ribosomal subunits. This procedure introduces only limited numbers of cleavable crosslinks between IF-3 and its nearest neighbors. The cleavable crosslinking reagent, 2-iminothiolane, was used to crosslink IF-3 in place to both 30S and 50S subunits. Ribosomal proteins S9/S11, S12, L2, L5 and L17 were found, by this approach, to be in close proximity to the factor in purified IF-3-subunit complexes. In addition, IF-3 was shown to alter the ultraviolet absorbance spectrum of E. coli 70S ribosomes at 10 mM Mg2+. The magnitude of the observed difference spectrum at a constant IF-3/ribosome ratio of 1.0, is linearly dependent upon ribosome concentration over the range 5 nM - 55 nM. Titration experiments indicated that the observed effect is maximal at an IF-3/ribosome ratio of approximately 1.0. These results are taken to indicate a conformational change in the 70S ribosome induced by IF-3.     

Consequences of a specific cleavage in situ of 16-S ribosomal RNA for polypeptide chain initiation.

The effect of bacteriocin (cloacin DF13) treatment of Escherichia coli ribosomes on initiation of protein synthesis has been studied in detail. In agreement with our previous findings [Baan et al. (1976) Proc. Natl Acad. Sci. U.S.A. 73, 702--706] it is shown that 70-S initiation complexes can be formed with cloacin-treated ribosomes, but that the initiation factor IF-1 does not function properly. The following pleiotropic effects of this factor have been studied: (a) the acceleration of ribosomal subunit exchange with 70-S couples; (b) the stimulation of the IF-3-mediated dissociation of 70-S ribosomes; (c) the stimulation of 30-S initiation complex formation; (d) the enhancement of the rate of release of IF-2 from 70-S initiation complexes. The effects (a) and (b) are virtually abolished after cleavage of 16-S rRNA. The effect (d) is only partially reduced whereas effect (c) seems to be unimpaired. It is concluded that 70-S initiation complex formation with cloacin-treated ribosomes suffers from improper functioning of IF-1 in the generation of active subunits from 70-S tight couples. This is the only effect on initiation. It can be compensated for by adding more IF-3. The data provide functional evidence that 16-S rRNA is involved in ribosomal subunit interaction.     

Immunological characterization of the complex forms of chloroplast translational initiation factor 2 from Euglena gracilis.

Euglena gracilis chloroplast translational initiation factor 2 (IF-2chl) occurs in several complex forms ranging in molecular mass from 200 to 800 kDa. Subunits of 97 to greater than 200 kDa have been observed in these preparations. Two monoclonal antibodies were prepared against the 97-kDa subunits of IF-2chl. Both of these antibodies recognize all of the higher molecular mass forms of this factor, suggesting that these subunits are closely related. Gel filtration chromatography indicates that the higher molecular mass subunits of IF-2chl are present in the higher molecular mass complexes, whereas the smaller subunits are present in the 200-400 kDa forms of IF-2chl. Probing extracts of light-induced and dark-grown cells with the antibodies indicates that the light induction of this chloroplast factor results from the synthesis of new polypeptide rather than from the activation of an inactive precursor form of the protein. Both the higher and lower molecular mass subunits of IF-2chl are present in 30 S initiation complexes as indicated by Western analysis. The binding of IF-2chl to chloroplast 30 S ribosomal subunits requires the presence of GTP, but does not require fMet-tRNA, messenger RNA, or other initiation factors. Neither polyclonal nor monoclonal antibodies against E. gracilis IF-2chl cross-react with Escherichia coli IF-2 or with animal mitochondrial IF-2.     

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+2 dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+2 (translationally inactive) and high Mg+2 (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit.     

Isolation of ribosomal subparticles from human placenta containing intact rRNA and determination of the functional activity of the 80S ribosome

The method for isolation of human placenta ribosomal subunits containing intact rRNA has been determined. The method uses fresh unfrozen placenta. Activity of 80S ribosomes obtained via reassociation of 40S and 60S subunits in non-enzymatic poly(U)-mediated Phe-tRNAPhe binding, was near 75% (maximal [14C]Phe-tRNA(Phe) binding was 1.5 mol Phe-tRNA(Phe) per mol of 80S ribosomes). Activity of 80S ribosomes with damaged rRNA isolated from frozen placenta was 2 times lower (the maximum level of poly(U)-dependent Phe-tRNA(Phe) binding was 0.7 mol per mol of ribosomes). The activity 80S ribosomes in poly(U)-mediated synthesis of polyphenylalanine was determined by using fractionated ("ribosomeless") protein synthesising system from rabbit reticulocytes. In this system up to the 50 mol of Phe residues per mol of 80S ribosomes are incorporated in acid insoluble fraction in 1 hour, at 37 degrees C. The obtained level of [14C]phenylalanine incorporation is three times as much as the amount of Phe residues observed for the ribosomal subunits, isolated from frozen placenta.     

The mechanism of action of initiaton factor 3 in protein synthesis,I. Studies on ribosomes crosslinked with dimethylsuberimidate

The mechanism of action of chain initiation factor 3 in translation was examined by using $\text{E. coli}$ 70S ribosomes which were covalently crosslinked with dimethylsuberimidate. Crosslinked ribosomes were inactive in AUG-dependent fMet-tRNA binding, and were not stimulated by IF-3 in poly(U) translation. IF-3 is known to be required for maximal rates of amino acid incorporation with synthetic polynucleotides at 18 mM Mg²⁺. A direct interaction of IF-3 with 70S ribosomes was demonstrated by crosslinking ¹⁴C-labeled IF-3 to 70S ribosomes. The labeled factor was also crosslinked to 30S and 50S ribosomal subunits. A model is presented proposing the mechanism of action of IF-3 on 70S ribosomes.     

Initial rate kinetic analysis of the mechanism of initiation complex formation and the role of initiation factor IF-3.

Initial rate kinetics of the formation of ternary complexes of Escherichia coli 30S ribosomal subunits, poly(uridylic acid), and N-acetylphenylalanyl transfer ribonucleic acid in the presence and in the absence of IF-3 are consistent with the hypothesis that the ternary complex is formed through a random order of addition of polynucleotide and aminoacyl-tRNA to separate and independent binding sites on the 30S ribosomes. The transformation of an intermediate into a stable ternary complex which probably entails a rearrangement of the ribosome structure leading to a codon-anticodon interaction represents the rate-limiting step in the formation of the ternary complex. The rate constant of this transformation, as well as the association constants for the formation of the 30S-poly(U) and 30S-N-AcPhe-tRNA binary complexes, are enhanced by the presence of IF-3 which acts as a kinetic effector on reactions which are intrinsic properties of the 30S ribosome. The IF-3-induced modification of these kinetic parameters of the 30S ribosomal subunit can per se explain the effect of IF-3 on protein synthesis without invoking a specific action at the level of the mRNA-ribosome interaction. This seems to be confirmed by the finding that IF-3 can stimulate several-fold the formation of a ternary complex even if one by-passes the ribosome-template binding step by starting with a covalent 30S-polynucleotide binary complex. Furthermore, the above-mentioned changes induced by IF-3 appear to be compatible with the previously proposed idea that the binding of the factor modifies the conformation of the 30S subunit. The random order of addition of substrates determined for the 30S-N-AcPhe-tRNA-poly(U) model system was found to be valid also for the more physiological 30S initiation complex containing poly(A,U.G) and (fMet-tRNA formed at low Mg2+ concentration in the presence of GTP and all three initiation factors.     

Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis

The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.