RESUMO
Eukaryotic protein synthesis begins with assembly of 48S initiation complexes at the initiation codon of mRNA, which requires at least seven initiation factors (eIFs). First, 43S preinitiation complexes comprising 40S ribosomal subunits, eIFs 3, 2, 1, and 1A, and tRNA(Met)(i) attach to the 5'-proximal region of mRNA and then scan along the 5' untranslated region (5'UTR) to the initiation codon. Attachment of 43S complexes is mediated by three other eIFs, 4F, 4A, and 4B, which cooperatively unwind the cap-proximal region of mRNA and later also assist 43S complexes during scanning. We now report that these seven eIFs are not sufficient for efficient 48S complex formation on mRNAs with highly structured 5'UTRs, and that this process requires the DExH-box protein DHX29. DHX29 binds 40S subunits and hydrolyzes ATP, GTP, UTP, and CTP. NTP hydrolysis by DHX29 is strongly stimulated by 43S complexes and is required for DHX29's activity in promoting 48S complex formation.
Assuntos
Regiões 5' não Traduzidas , Biossíntese de Proteínas , RNA Helicases/metabolismo , RNA Mensageiro/genética , Animais , Células HeLa , Humanos , Coelhos , Reticulócitos/metabolismoRESUMO
Platelets are anucleate and mostly ribosome-free cells within the bloodstream, derived from megakaryocytes within bone marrow and crucial for cessation of bleeding at sites of injury. Inherited thrombocytopenias are a group of disorders characterized by a low platelet count and are frequently associated with excessive bleeding. SLFN14 is one of the most recently discovered genes linked to inherited thrombocytopenia where several heterozygous missense mutations in SLFN14 were identified to cause defective megakaryocyte maturation and platelet dysfunction. Yet, SLFN14 was recently described as a ribosome-associated protein resulting in rRNA and ribosome-bound mRNA degradation in rabbit reticulocytes. To unveil the cellular function of SLFN14 and the link between SLFN14 and thrombocytopenia, we examined SLFN14 (WT/mutants) in in vitro models. Here, we show that all SLFN14 variants colocalize with ribosomes and mediate rRNA endonucleolytic degradation. Compared to SLFN14 WT, expression of mutants is dramatically reduced as a result of post-translational degradation due to partial misfolding of the protein. Moreover, all SLFN14 variants tend to form oligomers. These findings could explain the dominant negative effect of heterozygous mutation on SLFN14 expression in patients' platelets. Overall, we suggest that SLFN14 could be involved in ribosome degradation during platelet formation and maturation.
Assuntos
Endorribonucleases/genética , Endorribonucleases/metabolismo , RNA Ribossômico/metabolismo , Trombocitopenia/genética , Animais , Células Cultivadas , Células HEK293 , Humanos , Mutação de Sentido Incorreto , RNA Ribossômico 5,8S/análise , Coelhos , Ribossomos/química , Ribossomos/metabolismoRESUMO
Eukaryotic translation initiation is a complex process involving many components. eIF3 is a scaffold for multiple initiation factors and plays multiple roles in initiation, and DHX29 helicase enhances the formation of the 48S initiation complex on structured mRNAs. Because DHX29 is not a processive helicase, the mechanism underlying its activity is unclear. Here, we show that DHX29 establishes many points of contact with eIF3. In particular, the unique N terminus of DHX29 associates with the RNA recognition motif of eIF3b and the C terminus of the eIF3a subunits of eIF3, and the disruption of either contact impairs DHX29 activity. In turn, DHX29 has weak points of contact with mRNA in the 48S initiation complex, and the pathway taken by mRNA remains unchanged. These results exclude the direct role for this protein in unwinding. Thus, DHX29 and eIF3 cooperate in scanning on structured mRNAs. Our findings support previous genetic data on the role of eIF3 during scanning.
Assuntos
Fator de Iniciação 3 em Eucariotos/metabolismo , Biossíntese de Proteínas , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , HumanosRESUMO
After termination, eukaryotic 80S ribosomes remain associated with mRNA, P-site deacylated tRNA, and release factor eRF1 and must be recycled by dissociating these ligands and separating ribosomes into subunits. Although recycling of eukaryotic posttermination complexes (post-TCs) can be mediated by initiation factors eIF3, eIF1, and eIF1A (Pisarev et al., 2007), this energy-free mechanism can function only in a narrow range of low Mg(2+) concentrations. Here, we report that ABCE1, a conserved and essential member of the ATP-binding cassette (ABC) family of proteins, promotes eukaryotic ribosomal recycling over a wide range of Mg(2+) concentrations. ABCE1 dissociates post-TCs into free 60S subunits and mRNA- and tRNA-bound 40S subunits. It can hydrolyze ATP, GTP, UTP, and CTP. NTP hydrolysis by ABCE1 is stimulated by post-TCs and is required for its recycling activity. Importantly, ABCE1 dissociates only post-TCs obtained with eRF1/eRF3 (or eRF1 alone), but not post-TCs obtained with puromycin in eRF1's absence.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Ribossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Citidina Trifosfato/metabolismo , Fatores de Iniciação em Eucariotos/fisiologia , Guanosina Trifosfato/metabolismo , Humanos , Magnésio/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas/metabolismo , Uridina Trifosfato/metabolismoRESUMO
During eukaryotic translation initiation, the 43S preinitiation complex (43S PIC), consisting of the 40S ribosomal subunit, eukaryotic initiation factors (eIFs) and initiator tRNA scans mRNA to find an appropriate start codon. Key roles in the accuracy of initiation codon selection belong to eIF1 and eIF1A, whereas the mammalian-specific DHX29 helicase substantially contributes to ribosomal scanning of structured mRNAs. Here, we show that DHX29 stimulates the recognition of the AUG codon but not the near-cognate CUG codon regardless of its nucleotide context during ribosomal scanning. The stimulatory effect depends on the contact between DHX29 and eIF1A. The unique DHX29 N-terminal domain binds to the ribosomal site near the mRNA entrance, where it contacts the eIF1A OB domain. UV crosslinking assays revealed that DHX29 may rearrange eIF1A and eIF2α in key nucleotide context positions of ribosomal complexes. Interestingly, DHX29 impedes the 48S initiation complex formation in the absence of eIF1A perhaps due to forming a physical barrier that prevents the 43S PIC from loading onto mRNA. Mutational analysis allowed us to split the mRNA unwinding and codon selection activities of DHX29. Thus, DHX29 is another example of an initiation factor contributing to start codon selection.
Assuntos
RNA Helicases/fisiologia , Iniciação da Transcrição Genética , Códon de Iniciação , Escherichia coli , Fator de Iniciação 1 em Eucariotos/química , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Helicases/química , RNA Mensageiro/química , RNA de Transferência/químicaRESUMO
Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1's M domain contacts eRF3's GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1's stimulatory effect on eRF3's GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1's putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1.
Assuntos
Códon de Terminação/metabolismo , Modelos Moleculares , Fatores de Terminação de Peptídeos/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces , Trifosfato de Adenosina/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Ordem dos Genes , Humanos , Mutação , Fatores de Terminação de Peptídeos/genética , Ligação Proteica , Biossíntese de Proteínas/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Reprodutibilidade dos Testes , Ribossomos/metabolismo , Espalhamento a Baixo Ângulo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
48S initiation complex (48S IC) formation is the first stage in the eukaryotic translation process. According to the canonical mechanism, 40S ribosomal subunit binds to the 5'-end of messenger RNA (mRNA) and scans its 5'-untranslated region (5'-UTR) to the initiation codon where it forms the 48S IC. Entire process is mediated by initiation factors. Here we show that eIF5 and eIF5B together stimulate 48S IC formation influencing initiation codon selection during ribosomal scanning. Initiation on non-optimal start codons--following structured 5'-UTRs, in bad AUG context, within few nucleotides from 5'-end of mRNA and CUG start codon--is the most affected. eIF5-induced hydrolysis of eIF2-bound GTP is essential for stimulation. GTP hydrolysis increases the probability that scanning ribosomal complexes will recognize and arrest scanning at a non-optimal initiation codon. Such 48S ICs are less stable owing to dissociation of eIF2*GDP from initiator tRNA, and eIF5B is then required to stabilize the initiator tRNA in the P site of 40S subunit. Alternative model that eIF5 and eIF5B cause 43S pre-initiation complex rearrangement favoring more efficient initiation codon recognition during ribosomal scanning is equally possible. Mutational analysis of eIF1A and eIF5B revealed distinct functions of eIF5B in 48S IC formation and subunit joining.
Assuntos
Fator de Iniciação 5 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Ribossomos/metabolismo , Regiões 5' não Traduzidas , Códon de Iniciação , Fator de Iniciação 1 em Eucariotos/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 5 em Eucariotos/genética , Fatores de Iniciação em Eucariotos/genética , Guanosina Trifosfato/metabolismo , Mutação , RNA de Transferência de Metionina/metabolismoRESUMO
Turnover of mRNA is a critical step that allows cells to control gene expression. Endoribonucleases, enzymes cleaving RNA molecules internally, are some of the key components of the degradation process. Here we provide a detailed characterization of novel endoribonuclease SLFN14 purified from rabbit reticulocyte lysate. Schlafen genes encode a family of proteins limited to mammals. Their cellular function is unknown or incompletely understood. In reticulocytes, SLFN14 is strongly overexpressed, represented exclusively by the short form, all tethered to ribosomes, and appears to be one of the major ribosome-associated proteins. SLFN14 binds to ribosomes and ribosomal subunits in the low part of the body and cleaves RNA but preferentially rRNA and ribosome-associated mRNA. This results in the degradation of ribosomal subunits. This process is strictly Mg(2+)- and Mn(2+)-dependent, NTP-independent, and sequence nonspecific. However, in other cell types, SLFN14 is a full-length solely nuclear protein, which lacks ribosomal binding and nuclease activities. Mutational analysis revealed the ribosomal binding site and the aspartate essential for the endonucleolytic activity of protein. Only few endoribonucleases participating in ribosome-mediated processes have been characterized to date. Moreover, none of them are shown to be directly associated with the ribosome. Therefore, our findings expand the general knowledge of endoribonucleases involved in mammalian translation control.
Assuntos
Endorribonucleases/metabolismo , RNA Ribossômico/metabolismo , Coelhos/metabolismo , Reticulócitos/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Animais , Sítios de Ligação , Endorribonucleases/química , Endorribonucleases/genética , Mutação , Coelhos/genéticaRESUMO
Initiation codon selection in eukaryotes involves base-by-base inspection of the 5'-untranslated region of mRNA by scanning ribosomal 43S preinitiation complexes. We employed in vitro reconstitution to investigate factor requirements for this process and report that in the absence of eIF1 and DHX29, eIFs 4A, 4B and 4G promote efficient bypassing of stable stems by scanning 43S complexes and formation of 48S initiation complexes on AUG codons immediately upstream and downstream of such stems, without their unwinding. However, intact stems are not threaded through the entire mRNA Exit channel of the 40S subunit, resulting in incorrect positioning of mRNA upstream of the ribosomal P site in 48S complexes formed on AUG codons following intact stems, which renders them susceptible to dissociation by eIF1. In 48S complexes formed on AUG codons preceding intact stems, the stems are accommodated in the A site. Such aberrant complexes are destabilized by DHX29, which also ensures that mRNA enters the mRNA-binding cleft in a single-stranded form and therefore undergoes base-by-base inspection during scanning.
Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Animais , Sequência de Bases , Códon de Iniciação , Humanos , Biossíntese de Proteínas , RNA Mensageiro/genética , Subunidades Ribossômicas/genética , Subunidades Ribossômicas/metabolismo , Ribossomos/genética , Globinas beta/genéticaRESUMO
No-go decay (NGD) and non-stop decay (NSD) are eukaryotic surveillance mechanisms that target mRNAs on which elongation complexes (ECs) are stalled by, for example, stable secondary structures (NGD) or due to the absence of a stop codon (NSD). Two interacting proteins Dom34(yeast)/Pelota(mammals) and Hbs1, which are paralogues of eRF1 and eRF3, are implicated in these processes. Dom34/Hbs1 were shown to promote dissociation of stalled ECs and release of intact peptidyl-tRNA. Using an in vitro reconstitution approach, we investigated the activities of mammalian Pelota/Hbs1 and report that Pelota/Hbs1 also induced dissociation of ECs and release of peptidyl-tRNA, but only in the presence of ABCE1. Whereas Pelota and ABCE1 were essential, Hbs1 had a stimulatory effect. Importantly, ABCE1/Pelota/Hbs1 dissociated ECs containing only a limited number of mRNA nucleotides downstream of the P-site, which suggests that ABCE1/Pelota/Hbs1 would disassemble NSD complexes stalled at the 3'-end, but not pre-cleavage NGD complexes stalled in the middle of mRNA. ABCE1/Pelota/Hbs1 also dissociated vacant 80S ribosomes, which stimulated 48S complex formation, suggesting that Pelota/Hbs1 have an additional role outside of NGD.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Ribossomos/metabolismo , Animais , Eletroforese , Endonucleases , Escherichia coli , Vetores Genéticos/genética , Humanos , Técnicas In Vitro , Proteínas Nucleares , Coelhos , Proteínas Recombinantes/metabolismoAssuntos
Endorribonucleases/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Hemorragia/etiologia , Mutação , Alelos , Animais , Transtornos Plaquetários/complicações , Transtornos Plaquetários/genética , Plaquetas/metabolismo , Estudos de Associação Genética/métodos , Genótipo , HumanosRESUMO
Schlafen 14 (SLFN14) has recently been identified as an endoribonuclease responsible for cleaving RNA to regulate and inhibit protein synthesis. Early studies revealed that members of the SLFN family are capable of altering lineage commitment during T-cell differentiation by using cell-cycle arrest as a means of translational control by RNase activity. SLFN14 has been reported as a novel gene causing an inherited macrothrombocytopenia and bleeding in human patients; however, the role of this endoribonuclease in megakaryopoiesis and thrombopoiesis remains unknown. To investigate this, we report a CRISPR knock-in mouse model of SLFN14 K208N homologous to the K219N mutation observed in our previous patient studies. We used hematological analysis, in vitro and in vivo studies of platelet and erythrocyte function, and analysis of spleen and bone marrow progenitors. Mice homozygous for this mutation do not survive to weaning age, whereas heterozygotes exhibit microcytic erythrocytosis, hemolytic anemia, splenomegaly, and abnormal thrombus formation, as revealed by intravital microscopy, although platelet function and morphology remain unchanged. We also show that there are differences in erythroid progenitors in the spleens and bone marrow of these mice, indicative of an upregulation of erythropoiesis. This SLFN14 mutation presents distinct species-specific phenotypes, with a platelet defect reported in humans and a severe microcytic erythrocytosis in mice. Thus, we conclude that SLFN14 is a key regulator in mammalian hematopoiesis and a species-specific mediator of platelet and erythroid lineage commitment.
Assuntos
Plaquetas , Endorribonucleases/genética , Eritropoese , Animais , Linhagem da Célula/genética , Eritropoese/genética , Heterozigoto , Humanos , Camundongos , MutaçãoRESUMO
Taking control of the cellular apparatus for protein production is a requirement for virus progression. To ensure this control, diverse strategies of cellular mimicry and/or ribosome hijacking have evolved. The initiation stage of translation is especially targeted as it involves multiple steps and the engagement of numerous initiation factors. The use of structured RNA sequences, called Internal Ribosomal Entry Sites (IRES), in viral RNAs is a widespread strategy for the exploitation of eukaryotic initiation. Using a combination of electron cryo-microscopy (cryo-EM) and reconstituted translation initiation assays with native components, we characterized how a novel IRES at the 5'-UTR of a viral RNA assembles a functional initiation complex via an uAUG intermediate. The IRES features a novel extended, multi-domain architecture, that circles the 40S head. The structures and accompanying functional data illustrate the importance of 5'-UTR regions in translation regulation and underline the relevance of the untapped diversity of viral IRESs.
Assuntos
Dicistroviridae , Fator de Iniciação 3 em Eucariotos/ultraestrutura , Sítios Internos de Entrada Ribossomal , Modelos Moleculares , RNA Viral/ultraestrutura , Regiões 5' não Traduzidas , Animais , Microscopia Crioeletrônica , Fator de Iniciação 3 em Eucariotos/química , Fator de Iniciação 3 em Eucariotos/metabolismo , Humanos , Biossíntese de Proteínas/fisiologia , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Mensageiro/ultraestrutura , RNA Viral/química , RNA Viral/metabolismo , Subunidades Ribossômicas/química , Subunidades Ribossômicas/metabolismo , Subunidades Ribossômicas/ultraestruturaRESUMO
Co-opting the cellular machinery for protein production is a compulsory requirement for viruses. The Cricket Paralysis Virus employs an Internal Ribosomal Entry Site (CrPV-IRES) to express its structural genes in the late stage of infection. Ribosome hijacking is achieved by a sophisticated use of molecular mimicry to tRNA and mRNA, employed to manipulate intrinsically dynamic components of the ribosome. Binding and translocation through the ribosome is required for this IRES to initiate translation. We report two structures, solved by single particle electron cryo-microscopy (cryoEM), of a double translocated CrPV-IRES with aminoacyl-tRNA in the peptidyl site (P site) of the ribosome. CrPV-IRES adopts a previously unseen conformation, mimicking the acceptor stem of a canonical E site tRNA. The structures suggest a mechanism for the positioning of the first aminoacyl-tRNA shared with the distantly related Hepatitis C Virus IRES.
Assuntos
Dicistroviridae/genética , Hepacivirus/genética , Sítios Internos de Entrada Ribossomal/genética , Mimetismo Molecular/genética , RNA de Transferência/genética , RNA Viral/genética , Homologia de Sequência do Ácido Nucleico , Microscopia Crioeletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , RNA de Transferência/química , RNA Viral/química , Ribossomos/metabolismoRESUMO
Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that is responsible for the final step in initiation, which involves the displacement of initiation factors from the 40S ribosomal subunit in initiation complexes and its joining with the 60S subunit. Hydrolysis of eIF5B-bound GTP is not required for its function in subunit joining but is necessary for the subsequent release of eIF5B from assembled 80S ribosomes. Here we investigated the kinetics of guanine nucleotide binding to eIF5B by a fluorescent stopped-flow technique using fluorescent mant derivatives of GTP and GDP and of the GTP analogues GTPgammaS and GMPPNP. The affinity of eIF5B for mant-GTP (Kd approximately 14-18 microM) was approximately 7-fold less than for mant-GDP (Kd approximately 2.3 microM), and both guanine nucleotides dissociated rapidly from eIF5B (k-1mant-GTP approximately 22-28 s-1, k-1mant-GDP approximately 10-14 s-1). These properties of eIF5B suggest a rapid spontaneous GTP/GDP exchange on eIF5B and are therefore consistent with it having no requirement for a special guanine nucleotide exchange factor. The affinity of eIF5B for mant-GTPgammaS was about 2 times lower (Kd approximately 6.9 microM) and for mant-GMPPNP 1.5 times higher (Kd approximately 25.7 microM) than for mant-GTP, indicating that eIF5B tolerates modifications of the triphosphate moiety well.
Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Animais , Nucleotídeos de Guanina/metabolismo , Cinética , CoelhosRESUMO
Eukaryotic translation termination is mediated by two release factors: eRF1 recognizes stop codons and triggers peptidyl-tRNA hydrolysis, whereas eRF3 accelerates this process in a GTP-dependent manner. Here we report kinetic analysis of guanine nucleotide binding to eRF3 performed by fluorescence stopped-flow technique using GTP/GDP derivatives carrying the fluorescent methylanthraniloyl (mant-) group, as well as thermodynamic analysis of eRF3 binding to unlabeled guanine nucleotides. Whereas the kinetics of eRF3 binding to mant-GDP is consistent with a one-step binding model, the double-exponential transients of eRF3 binding to mant-GTP indicate a two-step binding mechanism, in which the initial eRF3.mant-GTP complex undergoes subsequent conformational change. The affinity of eRF3 for GTP (K(d), approximately 70 microM) is about 70-fold lower than for GDP (K(d), approximately 1 microM) and both nucleotides dissociate rapidly from eRF3 (k(-1)(mant-GDP) approximately 2.4 s(-1); k(-2)(mant-GTP) approximately 3.3 s(-1)). Whereas not influencing eRF3 binding to GDP, association of eRF3 with eRF1 at physiological Mg(2+) concentrations specifically changes the kinetics of eRF3/mant-GTP interaction and stabilizes eRF3.GTP binding by two orders of magnitude (K(d) approximately 0.7 microM) due to lowering of the dissociation rate constant approximately 24-fold (k(-1)(mant-GTP) approximately 0.14s(-1) approximately 0.14 s(-1)). Thus, eRF1 acts as a GTP dissociation inhibitor (TDI) for eRF3, promoting efficient ribosomal recruitment of its GTP-bound form. 80 S ribosomes did not influence guanine nucleotide binding/exchange on the eRF1 x eRF3 complex. Guanine nucleotide binding and exchange on eRF3, which therefore depends on stimulation by eRF1, is entirely different from that on prokaryotic RF3 and unusual among GTPases.
Assuntos
Guanosina Difosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Fatores de Terminação de Peptídeos/metabolismo , ortoaminobenzoatos/metabolismo , Animais , Sítios de Ligação , Transferência Ressonante de Energia de Fluorescência , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Cinética , Fatores de Terminação de Peptídeos/química , Coelhos , Termodinâmica , ortoaminobenzoatos/químicaRESUMO
Eukaryotic initiation factor (eIF) 1 maintains the fidelity of initiation codon selection and enables mammalian 43S preinitiation complexes to discriminate against AUG codons with a context that deviates from the optimum sequence GCC(A/G)CCAUGG, in which the purines at (-)3 and (+)4 positions are most important. We hypothesize that eIF1 acts by antagonizing conformational changes that occur in ribosomal complexes upon codon-anticodon base-pairing during 48S initiation complex formation, and that the role of (-)3 and (+)4 context nucleotides is to stabilize these changes by interacting with components of this complex. Here we report that U and G at (+)4 both UV-cross-linked to ribosomal protein (rp) S15 in 48S complexes. However, whereas U cross-linked strongly to C(1696) and less well to AA(1818-1819) in helix 44 of 18S rRNA, G cross-linked exclusively to AA(1818-1819). U at (-)3 cross-linked to rpS5 and eIF2alpha, whereas G cross-linked only to eIF2alpha. Results of UV cross-linking experiments and of assays of 48S complex formation done using alpha-subunit-deficient eIF2 indicate that eIF2alpha's interaction with the (-)3 purine is responsible for recognition of the (-)3 context position by 43S complexes and suggest that the (+)4 purine/AA(1818-1819) interaction might be responsible for recognizing the (+)4 position.