The arrangement of ribosomes in ribosome tetramers from hypothermic chick embryos

1. Ribosomes and the tetramer arrangement peculiar to the tissues of chick embryos exposed to low temperatures were separated by sucrose-density-gradient centrifugation, and the effects of variation of the concentrations of Mg(2+), Ca(2+) and K(+) studied. 2. Lowering of the Mg(2+) concentration from standard buffer conditions caused a reversible dissociation of tetramers into monomers and of these into subunits. 3. Ca(2+) replaced Mg(2+) in causing the re-formation of tetramers and monomers from subunits after dissociation in low Mg(2+) concentrations. 4. Ca(2+) also caused an almost complete conversion of monomers into dimers in the presence of Mg(2+). 5. The effect of Ca(2+) on the formation of dimers was abolished by pretreatment of the ribosomes with ribonuclease, but the re-formation of tetramers was unaffected. 6. Increase of the K(+) concentration from that of the standard buffer caused dissociation of monomers and dimers into subunits. 7. Raised K(+) concentration also caused a stepwise alteration of the tetramer from a particle with a sedimentation coefficient of 197S, which constitutes the bulk of the tetramer at low K(+) concentrations, first to a 184S peak and finally to material with a sedimentation coefficient of about 155S. 8. The implications of these results on hypotheses of the arrangement of the individual monomers in the tetramer are discussed and a new model for the structure is proposed.     

Stability of quaternary structure and mode of dissociation of fructosediphosphate aldolase isoenzymes

Using a highly sensitive "subunit exchange" assay, we have studied the relative strengths of interactions between different subunit types (A and C) of fructosediphosphate aldolase and have determined the mode of dissociation of aldolase tetramers in vitro. Interactions between C subunits within C4 tetramers were found to be considerably more resistant to disruption than were interactions between A subunits in A4 tetramers with regard to increasing concentrations of H+, OH-, or urea. Slight dissociation of A4 was also observed in 1.2 M magnesium chloride. These observations suggest that the quaternary structure of aldolase C4 is inherently more stable than that of aldolase A4. Also, the symmetrical heterotetramer A2C2 was found to be more resistant to urea-mediated dissociation than was the aldolase A4 homotetramer; this observation suggests that, even when in heteromeric combination, C subunits have a stabilizing influence on the quaternary structure of aldolase tetramers. In no case did we find evidence for a stable dimeric intermediate in the dissociation of aldolase tetramers to monomers. These observations are considered in terms of the tetrahedral arrangement of subunits in the aldolase tetramer. The general applicability of the subunit exchange assay described here for studying the subunit structure and mode of dissociation of oligomeric enzymes is discussed.     

Evidence for the lack of subunit exchange between aldolase tetramers in vivo.

The results of a double isotope experiment using 3H- and 14C-labeled leucine as precursors of protein synthesis demonstrated that the aldolase C to A subunit transition which is associated with chick skeletal muscle development involves the preferential synthesis of different aldolase isoenzymes. This developmental system was used to test for subunit exchange between aldolase tetramers in vivo. In a second double isotope experiment, it was found that the 14C:3H ratios of A and C subunits derived from the same heterotetramer were essentially identical, while the isotope ratios of the same subunit type derived from different isoenzymes were considerably different. Had subunit exchange between the isoenzymes occurred, A subunits of a given heterotetramer would have been expected to have higher isotope ratios than the corresponding C subunits. Therefore, these data suggest that subunit exchange between aldolase tetramers does not occur in vivo, at least not in skeletal muscle to an appreciable extent. The results of the present study suggest that all aldolase tetramers are constructed at the time of the initial assembly of newly synthesized subunits, that is, "new" tetramers would not be generated by subunit exchange between already constructed tetramers. In addition, the present work suggests that the degradation of all four subunits of an aldolase tetramer are coupled inasmuch as the subunits would not be reincorporated into other tetramers. Thus, in contrast to some other proteins, it appears that the subunits of the aldolase tetramer turn over coordinately.     

Electron transfer kinetics between hemoglobin subunits

The kinetics for electron transfer have been measured for samples of hemoglobin valency hybrids with initially one type of subunit, alpha or beta, in the oxidized state. Incubation of these samples under anaerobic conditions tends to randomize the type of subunit that is oxidized. With a time coefficient of a few hours at pH 7, 25 degrees C, the Hb solution (0.1 mm heme) approaches a form with about 60% of beta chains reduced, indicating a faster transfer rate in the direction alpha to beta. There was no observable electron transfer for samples saturated with oxygen. The electron transfer occurs predominantly between deoxy and aquo-met subunits, both high spin species. Furthermore, electron transfer does not depend on the quaternary state of hemoglobin. Incubation of oxidized cross-linked tetramer Hb A with deoxy Hb S also displayed electron transfer, implying a mechanism via inter-tetramer collisions. A dependence on the overall Hb concentration confirms this mechanism, although a small contribution of transfer between subunits of the same tetramer cannot be ruled out. These results suggest that in vivo collisions between the Hb tetramers will be involved in the relative distribution of the methemoglobin between subunits in association with the reductase system present in the erythrocyte.     

Hybrid tetramers of porcine liver fructose-1,6-bisphosphatase reveal multiple pathways of allosteric inhibition

Fructose-1,6-bisphosphatase is a square planar tetramer of identical subunits, which exhibits cooperative allosteric inhibition of catalysis by AMP. Protocols for in vitro subunit exchange provide three of five possible hybrid tetramers of fructose-1,6-bisphosphatase in high purity. The two hybrid types with different subunits in the top and bottom halves of the tetramer co-purify. Hybrid tetramers, formed from subunits unable to bind AMP and subunits with wild-type properties, differ from the wild-type enzyme only in regard to their properties of AMP inhibition. Hybrid tetramers exhibit cooperative, potent, and complete (100%) AMP inhibition if at least one functional AMP binding site exists in the top and bottom halves of the tetramer. Furthermore, titrations of hybrid tetramers with AMP, monitored by a tryptophan reporter group, reveal cooperativity and fluorescence changes consistent with an R- to T-state transition, provided that again at least one functional AMP site exists in the top and bottom halves of the tetramer. In contrast, hybrid tetramers, which have functional AMP binding sites in only one half (top/bottom), exhibit an R- to T-state transition and complete AMP inhibition, but without cooperativity. Evidently, two pathways of allosteric inhibition of fructose-1,6-bisphosphatase are possible, only one of which is cooperative.     

Quaternary structure of pig liver phosphofructokinase

The quaternary structure of phosphofructokinase from pig liver has been studied by electron microscopy. Particles ranging in size from tetramers to long flexible chains of tetramers were commonly observed. Phosphofructokinase tetramers are square planar and approximately 110 A on a side; individual subunits are roughly spherical, with a mean radius of 28 A. Chains are formed by end-to-end association of tetramers rather than by tetramer stacking. The geometry of association implies that phosphofructokinase tetramers possess D2 symmetry, with distinct isologous bonding domains for dimer, tetramer, and chain formation.     

Dissociation of the ribulosebisphosphate-carboxylase large-subunit binding protein into dissimilar subunits

The ribulosebisphosphate-carboxylase large-subunit binding protein from Pisum sativum chloroplasts is an oligomer of two types of subunit with the composition alpha 6 beta 6. These two subunits are immunologically distinct, show different partial protease digestion patterns and have different amino-terminal sequences. Leaves of Hordeum vulgare also contain an oligomeric binding protein composed of equal amounts of two types of subunit. Treatment of either P. sativum stromal extracts or purified binding protein with ATP and Mg2+ ions causes the dissociation of the oligomeric form of the binding protein to the monomeric subunits. This effect is highly specific for ATP since CTP, UTP, GTP, ADP, AMP, cyclic AMP, NADPH and pyrophosphate do not cause dissociation.     

Oligomeric protein associations: transition from stochastic to deterministic equilibrium

Transfer of electronic excitation energy (sensitized fluorescence) between donor and acceptor fluorophores separately attached to dimer or tetramer proteins is used to demonstrate the exchange of subunits among the undissociated particles. In dimers subjected to a pressure that produces half-dissociation, the exchange occurs at a rate that approaches the rate of dissociation. In the tetramers of glyceraldehydephosphate dehydrogenase and lactate dehydrogenase at 0 degrees C, the times for subunit exchange are nearly 2 orders of magnitude, and at room temperature 5-10 times longer than the time required to reach the dissociation equilibrium. By application of a novel method, pressure is shown to preferentially increase the rate of dissociation in dimers and decrease the rate of association in tetramers. From these observations, we conclude that the tetramers constitute a heterogeneous population, the members of which are dissociated by pressure according to individual molecular properties that can be retained over periods of time much longer than the time for equilibration of the dissociation. The dissociation of dimers exhibits the characteristics of the classical stochastic chemical equilibria, while those of the tetramers, like the more complex protein aggregates, must already be considered similar to the deterministic mechanical equilibria of macroscopic bodies.     

Formation and carbon monoxide‐dependent dissociation of Allochromatium vinosum cytochrome c′ oligomers using domain‐swapped dimers

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c′ from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four‐helix bundle structure containing a covalently bound five‐coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer–monomer transition according to the absence and presence of CO. Herein, domain‐swapped dimeric AVCP was constructed and utilized to form a tetramer and high‐order oligomers. The X‐ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain‐swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain‐swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit–subunit interactions at the interfaces of the domain‐swapped dimer and monomer subunits in the tetramer were also similar to the subunit–subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain‐swapped dimer and two monomers upon CO binding. Without monomers, the domain‐swapped dimers formed tetramers, hexamers, and higher‐order oligomers in the absence of CO, whereas the oligomers dissociated to domain‐swapped dimers in the presence of CO, demonstrating that the domain‐swapped dimer maintains the CO‐induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer–monomer transition protein.     

Mode of interaction of phosphofructokinase with the erythrocyte membrane

Phosphofructokinase is known to associate with the human erythrocyte membrane both in vitro and in vivo. Such association activates the enzyme in vitro by relieving the allosteric inhibition imposed by ATP (Karadsheh, N.S., and Uyeda, K. (1977) J. Biol. Chem. 252, 7418-7420). We now demonstrate that ADP, ATP, and NADH, all of which are known to bind to the enzyme's adenine nucleotide activation site, are particularly potent in eluting the enzyme from the membrane. In addition, both inside-out red cell membrane vesicles and a 23-kDa fragment containing the amino terminus of the membrane protein, band 3, cause a slow, partial, and reversible inactivation of phosphofructokinase. The dependence of the residual phosphofructokinase activity on phosphofructokinase concentration demonstrates that inactivation occurs through the dissociation of active tetramers to inactive dimers. Dimers of phosphofructokinase associate with the membrane more avidly than tetramers. The kinetics of phosphofructokinase inactivation are consistent with the dissociation of tetramers in solution followed by the binding of dimers to the membrane. There is no indication of an association equilibrium between tetramers and dimers of phosphofructokinase bound to the membrane. Taken together, these results suggest that the amino-terminal segment of band 3 binds to the adenine nucleotide activation site, which is thought to be located in a cleft between the dimeric subunits of phosphofructokinase. As a result, band 3 not only rapidly activates the phosphofructokinase tetramer but also slowly inactivates the enzyme by preferentially binding its dissociated subunits.     

High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli.

The collagen prolyl 4-hydroxylases (C-P4Hs), enzymes residing within the lumen of the endoplasmic reticulum, play a central role in the synthesis of all collagens. The vertebrate enzymes are alpha(2)beta(2) tetramers in which the two catalytic sites are located in the alpha subunits, and protein disulfide isomerase serves as the beta subunit. All attempts to assemble an active C-P4H tetramer from its subunits in in vitro cell-free systems have been unsuccessful, but assembly of a recombinant enzyme has been reported in several cell types by coexpression of the two types of subunit. An active type I C-P4H tetramer was obtained here by periplasmic expression in Escherichia coli strains BL21 and RB791. Further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E. coli strain resulted in large amounts of human type I C-P4H tetramer. The specific activity of the C-P4H tetramer purified from the cytoplasmic expression was within the range of values reported for human type I C-P4H isolated as a nonrecombinant enzyme or produced in the endoplasmic reticulum of insect cells, but the expression level, about 25 mg/l in a fermenter, is about 5-10 times that obtained in insect cells. The enzyme expressed in E. coli differed from those present in vivo and those produced in other hosts in that it lacked the N glycosylation of its alpha subunits, which may be advantageous in crystallization experiments.     

Construction of molecular assemblies via docking: modeling of tetramers with D2 symmetry

Comparative modeling methods are commonly used to construct models of homologous proteins or oligomers. However, comparative modeling may be inapplicable when the number of subunits in a modeled oligomer is different than in the modeling template. Thus, a dimer cannot be a template for a tetramer because a new monomer-monomer interface must be predicted. We present in this study a new prediction approach, which combines protein-protein docking with either of two tetramer-forming algorithms designed to predict the structures of tetramers with D2 symmetry. Both algorithms impose symmetry constraints. However, one of them requires identification of two of the C2 dimers within the tetramer in the docking step, whereas the other, less demanding algorithm, requires identification of only one such dimer. Starting from the structure of one subunit, the procedures successfully reconstructed 16 known D2 tetramers, which crystallize with either a monomer, a dimer or a tetramer in the asymmetric unit. In some cases we obtained clusters of native-like tetramers that differ in the relative rotation of the two identical dimers within the tetramer. The predicted structural pliability for concanavalin-A, phosphofructokinase, and fructose-1,6-bisphosphatase agrees semiquantitatively with the observed differences between the several experimental structures of these tetramers. Hence, our procedure identifies a structural soft-mode that allows regulation via relative rigid-body movements of the dimers within these tetramers. The algorithm also predicted three nearly correct tetramers from model structures of single subunits, which were constructed by comparative modeling from subunits of homologous tetrameric, dimeric, or hexameric systems.     

The role of ribosome recycling factor in dissociation of 70S ribosomes into subunits

Protein synthesis is initiated on ribosomal subunits. However, it is not known how 70S ribosomes are dissociated into small and large subunits. Here we show that 70S ribosomes, as well as the model post-termination complexes, are dissociated into stable subunits by cooperative action of three translation factors: ribosome recycling factor (RRF), elongation factor G (EF-G), and initiation factor 3 (IF3). The subunit dissociation is stable enough to be detected by conventional sucrose density gradient centrifugation (SDGC). GTP, but not nonhydrolyzable GTP analog, is essential in this process. We found that RRF and EF-G alone transiently dissociate 70S ribosomes. However, the transient dissociation cannot be detected by SDGC. IF3 stabilizes the dissociation by binding to the transiently formed 30S subunits, preventing re-association back to 70S ribosomes. The three-factor-dependent stable dissociation of ribosomes into subunits completes the ribosome cycle and the resulting subunits are ready for the next round of translation.     

Electrophoretic mobilities and diffusion coefficients of hemoglobin at high pH.

Diffusion studies by photon correlation of scattered laser light confirm the dissociation of the tetrameric form of human carboxyhemoglobin to dimers above pH 10 and provide new estimates of the subunit dissociation equilibrium constants in this pH range. Electrophoretic light-scattering experiments under the same conditions reveal that the electrophoretic mobilities of tetramers and dimers are indistinguishable to within instrumental resolution (ca. 7% in these experiments). The data imply an increase of the electrical charge on the dimer of at least 2.8 to 4.4 net negative charges upon dissociation. Mechanisms for the accumulation of negative charge by the dimer upon dissociation of the tetramer are proposed.     

Transthyretin slowly exchanges subunits under physiological conditions: A convenient chromatographic method to study subunit exchange in oligomeric proteins

Transthyretin (TTR) subunits were labeled with a charge-modifying tag to evaluate the possibility of subunit exchange between tetramers under physiological conditions. Starting with a mixture of two TTR homotetramers, one having all subunits tagged at the N termini and the other composed of untagged subunits, heterotetramer formation as a function of time and temperature was evaluated using ion exchange chromatography. The data indicate that the subunit exchange can occur under native conditions at physiological pH in vitro, albeit slowly. Wild-type TTR exchanges subunits on a timescale of days at 37 degrees C and on a timescale of hours at 4 degrees C. The familial amyloid polyneuropathy-associated variant V30M exchanges subunits at the same rate as wild-type TTR at 4 degrees C but slower and less efficiently at 37 degrees C. Small molecule tetramer stabilizers abolish TTR subunit exchange, supporting a dissociative mechanism.     

Hierarchy of globin complexes. The quaternary structure of the extracellular chlorocruorin of Eudistylia vancouverii.

The molecular dimensions of the extracellular, hexagonal bilayer chlorocruorin of the polychaete Eudistylia vancouverii, determined by scanning transmission electron microscopy (STEM) of negatively stained specimens, were diameter of 27.5 nm and height of 18.5 nm. STEM mass measurements of unstained, freeze-dried specimens provided a molecular mass (Mm) of 3480 +/- 225 kDa. The chlorocruorin had no carbohydrate and its iron content was 0.251 +/- 0.021 wt%, corresponding to a minimum Mm of 22.4 kDa. Mass spectra and nuclear magnetic resonance spectra of the prosthetic group confirmed it to be protoheme IX with a formyl group at position 3. SDS/polyacrylamide gel electrophoresis, reversed-phase chromatography and N-terminal sequencing suggested that the chlorocruorin consists of at least three chains of approximately 30 kDa and five chains of approximately 16 kDa; the two types of subunits occur in the ratio 0.26:0.74(+/- 0.08). Complete dissociation of the chlorocruorin at neutral pH in the presence of urea or guanidine hydrochloride, followed by gel filtration, produced elution profiles consisting of three peaks, B, C and D. Fractions B and C consisted of the approximately 16 kDa chains and fraction D consisted of the approximately 30 kDa subunits. Mass measurements of particles in STEM images of unstained, freeze-dried fractions B and C provided Mm of 208 +/- 23 kDa and 65 +/- 12 kDa, respectively, in agreement with 191 +/- 13 kDa and 67 +/- 5 kDa obtained by gel filtration. Particles with Mm = 221 +/- 21 kDa were also observed in STEM images of unstained, freeze-dried chlorocruorin. These results imply that the chlorocruorin structure, in addition to the approximately 30 kDa linker subunits that have 0.26 to 0.47 heme groups/chain, comprises approximately 65 kDa tetramers and approximately 200 kDa dodecamers (trimers of tetramers) of globin chains. The stoichiometry of the tetramer and linker subunits calculated from molar amino acid compositions was 34 +/- 4 and 43 +/- 9. The complete dissociation of the chlorocruorin was accompanied by a 50 to 75% loss of the 55 +/- 14 Ca2+/mol protein, and was decreased to approximately 35% by the presence of 10 to 25 mM-Ca2+. Reassociation of dissociated chlorocruorin was maximal in the presence of 2.5 to 5 mM-Ca2+. The dodecamer and/or tetramer subunits in the absence or presence of Ca2+ exhibited very limited (less than 10%) reassociation into hexagonal bilayer structures, only in the presence of the linker subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Catalytically Active Monomers of E. coli Glyceraldehyde-3-Phosphate Dehydrogenase

Monomeric forms of E. coli glyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane. Isolated monomers were shown to be catalytically active, exhibiting K _M values close to the parameters characteristic of the tetrameric forms. Like tetramers, isolated monomers did not use NADP7 as a coenzyme.     

Catalytically Active Monomers of E. coli Glyceraldehyde-3-Phosphate Dehydrogenase

Monomeric forms of E. coli glyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane. Isolated monomers were shown to be catalytically active, exhibiting K _M values close to the parameters characteristic of the tetrameric forms. Like tetramers, isolated monomers did not use NADP7 as a coenzyme.     

Effect of Sulfhydryl Reagents on the Ribosomes of Bacillus subtilis

The effect of various sulfhydryl reagents on the ribosomes of Bacillus subtilis was studied. The 70S ribosomes were completely dissociated into 30S and 50S subunits by appropriate concentrations of p-chloromercuribenzoic acid (PCMB) and 5,5'-dithio-bis-(2-nitro-benzoic acid). The N-ethylmaleimide and iodoacetamide failed to dissociate the ribosomes even at relatively high concentrations. The rate of dissociation of ribosomes by PCMB varied with the concentration of ribosomes. A progressive decrease in the rate of dissociation was observed as the concentration of ribosomes in the reaction mixture was increased. The PCMB-induced ribosomal subunits were unable to reassociate into 70S monomers unless they were dialyzed against buffer containing beta-mercaptoethanol. On the average, four molecules of PCMB per 70S ribosome and two molecules of PCMB per each 30S and 50S subunit were bound. The number of PCMB molecules bound per ribosome did not change with increasing concentrations of PCMB, even though higher concentrations of PCMB resulted in dissociation of ribosomes into subunits.     

Ribosome structure determined by electron microscopy of Escherichia coli small subunits,large subunits and monomeric ribosomes

Unique, three-dimensional structures have been determined for Escherichia coli small subunits, large Subunits and monomeric ribosomes by electron microscopy of ribosomes and subunits and of antibody-labeled ribosomes and subunits.Small subunits orient on the carbon substrate with their long axes parallel to the plane of the carbon. In these projections the subunit is divided into a onethird and a two-thirds portion by a region of accumulated negative stain similar to that observed in eukaryotic small subunits. Four characteristic views, or projections, are readily recognized and correspond to orientations of approximately ?40 °, 0 °, +50 ° and +110 ° about the long axis of the subunit. Three of these have been described (Lake et al., 1974a; Lake & Kahan, 1975). The two most distinctive views are a quasi-symmetric view (0 °) that is characterized by an approximate line of mirror symmetry that coincides with the long axis of the subunit, and an asymmetric view (110 °) that is characterized by a concave and a convex subunit boundary. In the asymmetric projection, a platform or ledge is viewed “face-on”. The platform is attached to the lower two-thirds of the subunit just below the one-third/two-thirds partition. It is separated from the upper one-third of the subunit at the level of the partition and above the partition it forms a cleft approximately 30 to 40 Å wide, which has been suggested as the site of the codon-anticodon interaction (Lake & Kahan, 1975).Four characteristic views are presented for the large subunit. The most prominent of these, the quasi-symmetric view (θ = 90 °, φ = 0 °), is distinguished by a central protuberance located on a line of approximate mirror symmetry. The central protuberance is surrounded by projecting features inclined at about 50 ° on both sides of it. The smaller of these projections is rod-like, about 40 Å wide and approximately 100 Å long. The feature projecting from the other side of the central protuberance is shorter, more blunt and wider than the rod-like appendage. In another view approximately orthogonal to the quasi-symmetric projection, the asymmetric projection (θ = 10 °, φ = 90 °), the subunit profile is distinguished by a convex lower edge and an upper boundary which is indented by a notch. The subunit is separated, in projection, by the notch into two unequal regions. The smaller region comprises about 20% of the total projected density and consists of the central protuberance and the rod-like appendage.The profiles observed in fields of monomeric 70 S ribosomes result from superpositions of the 30 S and 50 S profiles. Two major views are observed, an overlap and a non-overlap view, corresponding to whether or not the profile of the small subunit overlaps that of the large subunit in the 70 S profile. The small subunit is oriented in the monomeric ribosome so that the platform is in contact with the large subunit. The central protuberance of the large subunit overlaps part of the upper one-third of the small subunit in the overlap view of 70 S ribosomes, although in three dimensions they are probably separated by 30 to 50 Å. A region of the small subunit comprising the platform, the cleft and part of the upper one-third, suggested to be the approximate binding site of IF3 and IF2 (Lake & Kalian, 1975), is located at the interface between the large and small subunits, in a region of the small subunit that is close to, but probably not in physical contact with, the large subunit.