Structural basis for autoantibody recognition of phosphatidylserine-beta 2 glycoprotein I and apoptotic cells.

Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by beta 2 glycoprotein I (beta 2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-beta 2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells.

Diverse roles for the third complementarity determining region of the heavy chain (H3) in the binding of immunoglobulin Fv fragments to DNA, nucleosomes and cardiolipin.

Autoantibodies to DNA and chromatin employ junctional diversity and somatic mutations to generate or enhance antigen recognition. To define the role of diversity generating mechanisms in the etiology of autoantibodies to nuclear antigens, the heavy (H) chain of a murine autoantibody, 3H9, was used in its somatically mutated or germ-line form in conjunction with its own or with heterologous CDR3 (H3) domains. The resulting H chains were expressed together with the 3H9 light (L) chain as single-chain Fv (scFv) in Escherichia coli and assayed for binding to DNA, nucleosomes, or cardiolipin by enzyme-linked immunosorbent assay. All recombinant scFv exhibited nearly identical binding to cardiolipin. In contrast, the binding to nuclear antigens was drastically reduced by the reversion of mutations in 3H9 or the exchange of H3, such that only 3H9 itself bound strongly to single-stranded DNA, double-stranded DNA and nucleosomes. The results illustrate diverse interactions between a single combining site and different autoantigens. The analysis of these interactions suggests that the 3H9 VH domain, as encoded by the germ line, directs binding to cardiolipin, whereas structural determinants of H3, in concert with the remainder of the combining site, guide the maturation of antibody binding toward nuclear autoantigens.

Analysis of autoimmune bone marrow by antibody-phage display: somatic mutations and third complementarity-determining region arginines in anti-DNA gamma and kappa V genes.

OBJECTIVE: To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE). METHODS: A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of gamma, kappa, and lambda isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)- and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay. RESULTS: DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations. CONCLUSION: Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.

Tandem affinity tags for the purification of bivalent anti-DNA single-chain Fv expressed in Escherichia coli.

Antibodies to DNA define an important autospecificity that arises in systemic lupus erythematosus (SLE). To elucidate the molecular features that may explain the pathogenesis of SLE, a heterologous system for expression of cloned V genes is often desirable. Here, a single-chain Fv coding domain was constructed by using the heavy- and light-chain V genes of a high-affinity site-directed mutant of the murine anti-dsDNA autoantibody, 3H9. This scFv was joined in frame to the c-jun leucine zipper for dimerization, and to two affinity tags, domain B of the staphylococcal protein A and a pentahistidine peptide, for purification. Dimerization of the scFv was determined by size-exclusion chromatography. The yields of the scFv following affinity purification on IgG agarose or Ni-NTA agarose were compared, and the activities of the resulting protein fractions were determined. A two-step purification of periplasmic extracts on Ni-NTA agarose and IgG agarose, followed by elution with 3.5 M MgCl(2), yielded scFv with the highest specific activity. The final purified material bound DNA by ELISA, electrophoretic mobility shift assay, and immunofluorescence of fixed Hep-2 cells. Antibodies purified in this fashion should have applications in structure/function studies in which it is essential to generate highly purified antigen-combining sites.

Initiation of systemic autoimmunity and sequence specific anti-DNA autoantibodies.

Antibodies to double-stranded DNA (dsDNA) are a defining feature of Systemic Lupus Erythematosus (SLE). The molecular characterization of anti-dsDNA autoantibodies reveals that they are actively selected for binding to antigen. Evidence for antigen selection includes the use of suitable rearrangement products, the switching of IgM isotype to IgG, and the acquisition of somatic mutations that raise the affinity for dsDNA. Through a process of specificity maturation, anti-dsDNA antibodies can arise from anti-single stranded DNA (ssDNA) antibodies that also occur in nonautoimmune individuals. To clarify circumstances leading to the initiation of systemic autoimmunity, we compare features of immune responses to nucleic acids that operate before and after disease develops. Evidence indicating that anti-dsDNA antibodies bind with DNA sequence preference is highlighted to propose that sequence-specific anti-dsDNA antibodies may be induced by an infectious agent and in turn may extend the response to endogenous nuclear antigens. Thus, sequence-specific anti-dsDNA B cells may provide an important stimulus to break the tolerance to self.

Selection of recurrent V genes and somatic mutations in autoantibodies to DNA.

Antigen selection of autoantibodies to DNA results in the use of limited sets of immunoglobulin heavy and light chains, characteristic VDJ and VJ joining regions, and recurrent patterns of somatic mutations. In the past, we have used site-directed mutagenesis to examine the roles of two recurrent features of anti-DNA antibodies: VHCDR3 arginine and somatic mutations to arginine. We observed that in one prototypic anti-DNA antibody, 3H9, a suitable CDR3 conformation is essential for DNA binding and depends on arginine. In addition, arginines at any of five positions in CDR1, CDR2, or FWR3 of the heavy chain contribute contacts with the antigen. Here, we extend these studies and report that arginines at positions 52 and 58 improve relative DNA binding but that binding critically depends on the germline-encoded arginine at position 50. These observations provide a more detailed view of the anti-DNA combining site and suggest that structural features account, at least in part, for the recurrence of heavy chain variable regions in anti-DNA antibodies.

Chicken vitellogenin gene-binding protein, a leucine zipper transcription factor that binds to an important control element in the chicken vitellogenin II promoter, is related to rat DBP.

We screened a chicken liver cDNA expression library with a probe spanning the distal region of the chicken vitellogenin II (VTGII) gene promoter and isolated clones for a transcription factor that we have named VBP (for vitellogenin gene-binding protein). VBP binds to one of the most important positive elements in the VTGII promoter and appears to play a pivotal role in the estrogen-dependent regulation of this gene. The protein sequence of VBP was deduced from a nearly full length cDNA copy and was found to contain a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP binds to its target DNA site as a dimer. Moreover, VBP is a stable dimer free in solution. A data base search revealed that VBP is related to rat DBP. However, despite the fact that the basic/hinge regions of VBP and DBP differ at only three amino acid positions, the DBP binding site in the rat albumin promoter is a relatively poor binding site for VBP. Thus, the optimal binding sites for VBP and DBP may be distinct. Similarities between the VBP and DBP leucine zippers are largely confined to only four of the seven helical spokes. Nevertheless, these leucine zippers are functionally compatible and appear to define a novel subfamily. In contrast to the bZIP regions, other portions of VBP and DBP are markedly different, as are the expression profiles for these two genes. In particular, expression of the VBP gene commences early in liver ontogeny and is not subject to circadian control.

Mutational studies reveal a complex set of positive and negative control elements within the chicken vitellogenin II promoter.

The endogenous chicken vitellogenin II (VTGII) gene is transcribed exclusively in hepatocytes in response to estrogen. We previously identified two estrogen response elements (EREs) upstream of this gene. We now present an analysis of the VTGII promoter activated by these EREs in response to estrogen. Chimeric VTGII-CAT genes were cotransfected into LMH chicken hepatoma cells along with an estrogen receptor expression vector, and transient CAT expression was assayed after culturing the cells in the absence or presence of estrogen. An analysis of constructs bearing deletions downstream of the more proximal ERE indicated that promoter elements relevant to transcription in LMH cells extend to between -113 and -96. The relative importance of sequences within the VTGII promoter was examined by using 10 contiguous linker scanner mutations spanning the region from -117 to -24. Although most of these mutations compromised VTGII promoter function, one dramatically increased expression in LMH cells and also rendered the VTGII promoter capable of being activated by cis-linked EREs in fibroblasts cotransfected with an estrogen receptor expression vector. Gel retardation and DNase I footprinting assays revealed four factor-binding sites within this promoter. We demonstrate that three of these sites bind C/EBP, SP1, and USF (or related factors), respectively; the fourth site binds a factor that we denote TF-V beta. The biological relevance of these findings is suggested by the fact that three of these binding sites map to sites previously shown to be occupied in vivo in response to estrogen.

Ribosome binding to inosine-substituted mRNAs in the absence of ATP and mRNA factors.

Incubating ribosomes and eukaryotic initiation factor eIF3 with an inosine-substituted mRNA (where the mRNA secondary structure is strongly reduced) in the absence of ATP and other protein synthesis factors produces a 40 S ribosome.mRNA complex. When Met-tRNAMeti and eIF2 are added, a 60 S ribosome subunit attaches forming an 80 S ribosome.mRNA complex. ATP and the three mRNA factors, eIF4B, cap-site factor, and eIF4A, strongly stimulate the attachment of the 60 S subunit. In the absence of Met-tRNAMeti, the 60-S subunit does not attach, and adding ATP and the mRNA factors inhibits the accumulation of 40 S ribosome.inosine mRNA complexes. These results indicate that a 40 S ribosome, probably in a complex with eIF3, has an intrinsic capacity to attach to mRNA. Further, they suggest that Met-tRNAMeti may interact in a subsequent step to stabilize the 40 S ribosome.mRNA complex and allow the attachment of a 60 S ribosome subunit. Although seen most clearly with the inosine-substituted mRNAs, the 40 S ribosome reaction is also obtained with "guanosine" mRNA. A 40 S ribosome attaches to guanosine mRNA without ATP and mRNA factors when an incubation mixture containing ribosomes, eIF3, and mRNA is fixed with glutaraldehyde. In addition, a 40 S ribosome.guanosine mRNA complex can be obtained without glutaraldehyde in incubations containing ATP and the three mRNA factors in the absence of Met-tRNAMeti. The latter reaction is limited because of the instability of the 40 S ribosome.mRNA complex in the absence of Met-tRNA. Nevertheless, its authenticity is indicated by its full dependence upon ATP and the three mRNA factors. The lack of factor requirement for the formation of 40 S ribosome complexes with inosine-substituted mRNAs indicates that ATP and the three mRNA factors function primarily to unwind the secondary structure of a guanosine mRNA. Data relevant to a role for ATP in facilitating ribosome migration on an mRNA are also discussed.

Characterization of a phosphoenzyme intermediate in the reaction of phosphoglycolate phosphatase.

When 32P-glycolate and phosphoglycolate phosphatase from spinach are mixed, 32P is incorporated into acid precipitated protein. Properties that relate the phosphorylation of the enzyme to the phosphatase are: the Km value for P-glycolate is similar for protein phosphorylation and substrate hydrolysis; the 32P in the phosphoenzyme is diluted by unlabeled P-glycolate or the specific alternative substrate, ethyl-P; the activator Cl- enhances the effectiveness of ethyl-P as a substrate and as an inhibitor of the formation of 32P-enzyme; and 32P is lost from the enzyme when 32P-glycolate is consumed. The phosphorylated protein has a molecular weight of 34,000, which is half that of the native protein and is similar in size to the labeled band that is seen on sodium dodecyl sulfate-polyacrylamide gels. The enzyme-bound phosphoryl group appears to be an acylphosphate from its pH stability, being quite stable at pH 1, less stable at pH 5, and very unstable above pH 5. The bond is readily hydrolyzed in acid molybdate and it is sensitive to cleavage by hydroxylamine at pH 6.8. The demonstration of enzyme phosphorylation by 32P-glycolate resolves the dilemma presented by initial rate studies in which alternative substrates appeared to have different mechanisms (Rose, Z. B., Grove, D. S., and Seal, S. N. (1986) J. Biol. Chem. 261, 10996-11002).

Mechanism of activation by anions of phosphoglycolate phosphatases from spinach and human red blood cells.

Phosphoglycolate phosphatases from spinach and human red blood cells show a number of common features not often found in enzymes. Both enzymes are activated more than 50-fold by millimolar concentrations of Cl-. Other inorganic anions and a number of carboxylic acids also activate. Each enzyme has limited substrate specificity yet each hydrolyzes P-glycolate and ethyl-P with the same maximal velocity. L-P-lactate is only a good substrate for the red cell enzyme. With both enzymes initial rate data obtained by varying both the P-glycolate and Cl- give parallel line double reciprocal plots. Similar experiments with ethyl-P as substrate give intersecting lines with both enzymes. The likelihood that both classes of substrates are acting at the same site is strengthened by the results of inhibition studies with alternative substrates and the constancy of inhibition constants for glycolate with all substrates for a given enzyme. For each substrate the experimentally observed variation in V/Km with different activators is small, suggesting that the enzyme has an ordered mechanism with the phosphorylated substrate reacting first. A mechanism that is consistent with all of the data is presented.

A wheat germ cap-site factor functional in protein chain initiation.

Component C1 from wheat germ, a factor that functions in attaching ribosomes to mRNA, has been resolved into a fraction that does not bind to m7GDP-agarose (referred to as eIF4B) and one that binds and is eluted specifically by m7GDP. Both components are required for the attachment of ribosomes to [3H]methyl-labeled reovirus RNA and for the translation of a number of mRNAs, including the noncapped RNA of satellite tobacco necrosis virus. The component that binds to m7GDP-agarose, referred to as CSF (cap-site factor), contains primarily proteins of Mr 24,000, 26,000, and 75,000. Crosslinking studies with oxidized [3H]methyl-labeled reovirus RNA show that one of the lower molecular weight polypeptides of CSF interacts specifically with the 5'-cap of the mRNA in the absence of any other components. Incubation of component C1 and eIF4A in the presence of ATP results in the additional crosslinking of a 51- and a 65-kDa protein. In the absence of eIF4A, there is only the crosslinking of the lower molecular mass polypeptide (24 or 26 kDa). Attempts to reconstitute the C1 reaction with CSF and eIF4B result in a considerably diminished reaction. Crosslinking of eIF4A, however, is obtained in an incubation containing only CSF and eIF4A, suggesting that CSF may bring about an initial interaction of eIF4A with the 5' end of the mRNA.

Initiation factors eIF4A and C1 from wheat germ and the formation of mRNA X ribosome complexes.

The binding of ribosomes to mRNA is analyzed in a fractionated system from wheat germ with [3H]uridine-labeled poly(A)+ RNA prepared from germinating wheat embryos. The reaction requires factors eIF3, eIF4C, and eIF5; Met-tRNA and the Met-tRNA binding system; either GTP or GMP-PNP; ATP; and factors C1 and eIF4A. These requirements are identical to those previously found to be necessary for formation of ribosome X Met-tRNAMeti complexes, with the exception of ATP, and factors C1 and eIF4A. The function of factors C1 and eIF4A is therefore specifically related to the mRNA attachment reaction. The presence of GTP in the mRNA binding reaction results in the formation of 80 S ribosome complexes, while with GMP-PNP only 40 S ribosome complexes are formed. Ribosome binding to native reovirus RNA in the fractionated wheat germ system is similar to the reaction with poly(A)+ RNA, strongly requiring ATP and factors C1 and eIF4A. Binding to inosine-substituted reovirus RNA, however, is only partially dependent upon ATP, and both the ATP-dependent and the ATP-independent binding reactions strongly require factor C1 and are substantially stimulated by factor eIF4A. The ATP-independent reaction is inhibited by pm7GDP, has a strong requirement for Met-tRNAMeti, and the 40 S ribosome complex is stable to RNase. These results indicate that the ATP-independent binding of ribosomes to inosine-substituted reovirus RNA proceeds through the normal initiation process. They further suggest that neither factor C1 nor eIF4A function exclusively to unwind mRNA secondary structure. Since eIF4A is required for the ATP-independent binding to inosine mRNA, and at the same time interacts with ATP in the reaction with ATP-requiring mRNAs, this factor may have two roles in protein chain initiation, one related to the mRNA X ribosome interaction, and one related to the function of ATP.

Eukaryotic initiation factor 4A is the component that interacts with ATP in protein chain initiation.

Protein synthesis in a resolved homogenate of wheat germ requires ATP and eight factors functioning at the level of protein chain initiation. To identify the component(s) interacting with ATP, the different factors were treated with the ATP affinity analogue 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and tested for their function in protein synthesis. The activity of eukaryotic initiation factor 4A (eIF4A) was strongly curtailed, whereas all other factors were unaffected. At a concentration of 250 microM, AMP, ADP, and ATP protected eIF4A against FSBA inactivation, whereas at a concentration 50 microM, protection was afforded only by ATP. GTP did not protect at a concentration of 250 microM. In another approach, the substrate analogue 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) was found to inhibit protein synthesis in a manner, at least in part, competitive with ATP. Supplementing a TNP-ATP inhibited reaction with eIF4A substantially reversed the inhibition. Except for a small effect by factor C1, no reversal was obtained with any other component. Finally, a preincubation of ribosomes with ATP, mRNA, and eIF4A resulted in the formation of a complex capable of TNP-ATP-resistant amino acid incorporation. These data are interpreted to indicate that the primary interaction of ATP is with eIF4A. A model is proposed reconciling this conclusion with other observations relevant to the mRNA . ribosome attachment reaction.

Wheat germ eIF2 and CoeIF2. Resolution and functional characterization in in vitro protein synthesis.

A factor that binds Met-tRNAiMet in a ternary complex (eucaryotic initiation factor (eIF2) ) and an auxiliary component that strongly stimulates the activity of the binding factor (CoeIF2) have been purified from extracts of wheat germ. The molecular weights of the two factors, as determined by glycerol gradient centrifugation and sodium dodecyl sulfate gel electrophoresis, are 88,000 and 20,000, respectively. Incubation of wheat germ eIF2 with gamma-[32P]ATP and alpha-subunit kinase results in the phosphorylation of a Mr = 40,500 subunit, while no specific phosphorylations are found when the eIF2 is incubated with the beta-kinase. These data are interpreted to suggest that CoeIF2 is a monomer of Mr = 20,000 while eIF2 may be a dimer, containing subunits of Mr = 40,000 and 50,000. Both eIF2 and CoeIF2 are strongly required for in vitro amino acid polymerization, providing the first direct demonstration of the function of CoeIF2 in protein synthesis.

Fractionation and partial characterization of the protein synthesis system of wheat germ. I. Resolution of two elongation factors and five initiation factors.

Wheat germ S100 supernatant was resolved into 10 components, all of which are required for tobacco mosaic virus RNA-directed incorporation of amino acid into protein. Two of the components, C2b and C2a, are, respectively, elongation factors 1 and 2, having molecular weights of 51,000 and 72,000. A third factor (C2e) binds Met-tRNA in a GTP-requiring reaction and is absolutely required for the formation of both 40 S ribosome.Met-tRNAiMet and 80 S ribosome.Met-tRNAiMet complexes. This factor is considered to be eucaryotic initiation factor 2. Formation of 80 S ribosome Met-tRNAiMet complexes is augmented 2 to 3-fold by the simultaneous addition of an mRNA, factor C1, and fraction D2(a + b). Factor C1 also reverses the inhibition of translation by an increased concentration of monovalent cations and promotes the translation of a "competitively inhibited" mRNA. These observations suggest that factor C1 and either factor D2a and D2b (or both) function in mRNA attachment reactions. Factor C1 has a molecular weight of 115,000 and appears to be made up of two or three subunits. Factor D2b has been purified to essential homogeneity and has a molecular weight of 55,000. Its interaction with mRNA and its molecular weight suggest that this factor is the wheat germ equivalent of reticulocyte eucaryotic initiation factor 4A.

Fractionation and partial characterization of the protein synthesis system of wheat germ. II. Initiation factors D1 (eucaryotic initiation factor 3), D2c (eucaryotic initiation factor 5), and D2d (eucaryotic initiation factor 4C).

This report characterizes three components of the fractionated wheat germ protein synthesizing system, factors D1, D2c, and D2d. FActor D1 has a molecular weight of about 420,000 and is comprised of 7-10 components varying in molecular mass between 28,000 and 105,000 daltons. The factor is absolutely required for the formation of both 40-S ribosome.Met-tRNAiMet and 80 S ribosome.Met-tRNAiMet complexes except at low Mg2+ concentration, where significant 40 S ribosome complex formation can be obtained without D1, provided the reaction products are fixed with glutaraldehyde. Factor D1 can be shown to interact with the 40-S ribosome subunit in two ways: the change in sedimentation of the ribosome subunits that occurs when ribosomes are incubated with the factor under dissociating conditions and the prevention of the reformation of 80 S ribosomes by a prior incubation of ribosome subunits with the factor. Factor D2c has a molecular mass of about 100,000 daltons and is absolutely required for the conversion of 40 S ribosome.Met-tRNAiMet complexes to 80 S ribosome.Met-tRNAiMet. Factor D2d strongly augments the D2c-dependent formation of 80S ribosome.Met-tRNAiMet complexes, with the requirement for D2d most apparent when the complexes are fixed with glutaraldehyde, a treatment that labilizes the 80 S ribosome-bound Met-tRNAiMet. Addition of factor D2d to a 40 S ribosome.Met-tRNAiMet synthesizing system also increases the amount of complex obtained 2-5-fold. Consideration of these characteristics establishes components D1, D2c, and D2d as wheat germ eucaryotic initiation factors 3, 5, and 4C, respectively.

A heat-stable protein synthesis initiation factor from wheat germ.

A protein synthesis initiation factor of molecular weight 21,500 has been purified to homogeneity from extracts of wheat germ. The factor, referred to as D2d, is stable to heating for 5 min at 75 degrees C and loses less than 15% of its activity upon a similar treatment at 90 degrees C. In contrast, all other wheat germ protein synthesis factors are inactivated by heating for 5 min at 65 degrees C. Formation of 80 S ribosome . Met-tRNAiMet complexes from 40 S ribosome . Met-tRNAiMet complexes, as measured by the binding of [3H]Met-tRNAiMet, is almost completely dependent upon the presence of component D2d. The shift in sedimentation of the ribosome subunits to the 80 S ribosome region, however, occurs in the absence of factor D2d. Thus, the role of the factor is not to join the ribosomal subunits, but rather to keep the Met-tRNAiMet bound to the ribosome. The formation of 40 S ribosome . Met-tRNAiMet complexes is increased 2-fold by the presence of factor D2d. Fixing this complex with glutaraldehyde results in the binding of more Met-tRNAIMet than a nonfixed incubation in the presence of D2d. These results suggest that the primary interaction with the ribosomal subunit can occur in the absence of factor D2d, but that the presence of the factor stabilizes the ribosome-bound Met-tRNAiMet.

Formation of an 80 S methionyl-tRNA initiation complex with soluble factors from wheat germ.

Wheat germ supernatant is resolved into four fractions, C3alpha, C3beta, D and A, all of which are required in addition to elongation EF1 and EF2 for tobacco mosaic virus-RNA (TMV-RNA)-catalyzed amino acid polymerization. Fractions C3beta and D function in the binding of Met-tRNAiMet to the 40 S ribosomal subunit forming an unstable 40 S-Met-tRNA1Met complex that is detected in sucrose gradients only after fixation with glutaraldehyde. Fraction C3beta binds Met-tRNAiMet suggesting that a C3beta-Met-tRNAiMet complex may be an intermediate in the formation of the 40 S complex. GTP is required for the formation of both the C3beta-Met-tRNAiMet and the 40 S-Met-tRNAiMet complexes. In either reaction the nucleotide is partially replaceable by quanyl-5'-yl methylene diphosphonate (GMP-P (CH2) P) while ATP is inactive. When ATP, C3alpha, mRNA, and magnesium acetate (final concentration 3.6 mM) are added to an incubation reaction containing the 40 S-Met-tRNAiMet complex, a stable 80 S-Met-tRNAiMet complex is formed. Radioactive TM virus-RNA binds specifically to the 80 S-Met-tRNAi complex suggesting that mRNA is a component of this complex. C3beta and D, the fractions required in the formation of the 40 S-Met-tRNAiMet complex, are also required for formation of the 80 S-Met-tRNAiMet complex. In addition, unlabeled Met-trnaiMet does not compete significantly with preformed 40 S-Met-tRNAiMet in the 80 S complex-forming reaction. These observations suggest that the 40 S-Met-tRNAiMet complex is an intermediate in the 80 S reaction. The 80 S-Met-tRNAiMet product of the sequential reaction is reactive with puromycin and on this basis it is tentatively considered to be a functional initiation complex. The additional requirement of Fraction A for amino acid polymerization suggests, however, that the initiation process may be more complex.