Ribosome-binding Domain of Eukaryotic Initiation Factor-2 Kinase GCN2 Facilitates Translation Control

A family of protein kinases regulate translation initiation in response to cellular stresses by phosphorylation of eukaryotic initiation factor-2 (eIF-2). One family member from yeast, GCN2, contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic domain. It is thought that uncharged tRNA accumulating during amino acid starvation binds to the synthetase-related sequences and stimulates phosphorylation of the α subunit of eIF-2. In this report, we define another domain in GCN2 that functions to target the kinase to ribosomes. A truncated version of GCN2 containing only amino acid residues 1467 to 1590 can independently associate with the translational machinery. Interestingly, this region of GCN2 shares sequence similarities with the core of the double-stranded RNA-binding domain (DRBD). Substitutions of the lysine residues conserved among DRBD sequences block association of GCN2 with ribosomes and impaired the ability of the kinase to stimulate translational control in response to amino acid limitation. Additionally, as found for other DRBD sequences, recombinant protein containing GCN2 residues 1467–1590 can bind double-stranded RNAin vitro, suggesting that interaction with rRNA mediates ribosome targeting. These results indicate that appropriate ribosome localization of the kinase is an obligate step in the mechanism leading to translational control by GCN2. A family of protein kinases regulate translation initiation in response to cellular stresses by phosphorylation of eukaryotic initiation factor-2 (eIF-2). One family member from yeast, GCN2, contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic domain. It is thought that uncharged tRNA accumulating during amino acid starvation binds to the synthetase-related sequences and stimulates phosphorylation of the α subunit of eIF-2. In this report, we define another domain in GCN2 that functions to target the kinase to ribosomes. A truncated version of GCN2 containing only amino acid residues 1467 to 1590 can independently associate with the translational machinery. Interestingly, this region of GCN2 shares sequence similarities with the core of the double-stranded RNA-binding domain (DRBD). Substitutions of the lysine residues conserved among DRBD sequences block association of GCN2 with ribosomes and impaired the ability of the kinase to stimulate translational control in response to amino acid limitation. Additionally, as found for other DRBD sequences, recombinant protein containing GCN2 residues 1467–1590 can bind double-stranded RNAin vitro, suggesting that interaction with rRNA mediates ribosome targeting. These results indicate that appropriate ribosome localization of the kinase is an obligate step in the mechanism leading to translational control by GCN2. Targeting of proteins to different compartments in the cell is an important mechanism regulating protein function. Proteins can associate with organelles, membranes, or components in the soluble fraction of the cell, providing proteins access to substrates or regulatory ligands. GCN2 is a member of a family of protein kinases that regulate translation by phosphorylation of eukaryotic translation initiation factor-2 (eIF-2) 1The abbreviations used are: eIF-2, eukaryotic initiation factor-2; ds, double-stranded; DRBD, double-stranded RNA-binding domain; PAGE, polyacrylamide gel electrophoresis. (1Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996Google Scholar, 2Wek R.C. Trends Biochem. Sci. 1994; 19: 491-496Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 3Samuel C.E. J. Biol. Chem. 1993; 268: 7603-7606Abstract Full Text PDF PubMed Google Scholar, 4Hinnebusch A.G. J. Biol. Chem. 1997; 272: 21661-21664Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). Localization of GCN2 protein kinase to ribosomes appears to be a critical step leading to phosphorylation of eIF-2 in response to cellular stress. Phosphorylation of eIF-2 is a well characterized mechanism regulating eukaryotic protein synthesis. The eIF-2 is a three-subunit protein that couples with Met-tRNA i Met and participates in ribosomal selection of the start codon (5Merrick W.C. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-70Google Scholar). During this initiation process, GTP bound to eIF-2 is hydrolyzed to GDP. Phosphorylation of the α subunit of eIF-2 at serine 51 impedes recycling of eIF-2-GDP to the active form, eIF-2-GTP. Currently, three protein kinases that phosphorylate this regulated site of eIF-2 have been characterized and their cDNAs cloned (1Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1996Google Scholar, 2Wek R.C. Trends Biochem. Sci. 1994; 19: 491-496Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 3Samuel C.E. J. Biol. Chem. 1993; 268: 7603-7606Abstract Full Text PDF PubMed Google Scholar). Two of the proteins regulate protein synthesis in mammalian cells. The RNA-dependent protein kinase, PKR, participates in the antiviral defense mechanism mediated by interferon (6Proud C.G. Trends Biochem. Sci. 1995; 20: 217-256Abstract Full Text PDF PubMed Scopus (201) Google Scholar) and is also thought to function as a suppressor of cell proliferation and tumorigenesis (7Koromilas A.E. Roy S. Barber G.N. Katze M.G. Sonenberg N. Science. 1992; 257: 1685-1689Crossref PubMed Scopus (512) Google Scholar, 8Meurs E.F. Galabru J. Barber G.N. Katze M.G. Hovanessian A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 232-236Crossref PubMed Scopus (429) Google Scholar, 9Barber G.N. Jagus R. Meurs E.F. Hovanessian A.G. Katze M.G. J. Biol. Chem. 1995; 270: 17423-17428Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), and the heme-regulated inhibitor kinase, HRI, is expressed predominately in reticulocytes and bone marrow and couples the synthesis of globin, the principal translation product in these tissues, to hemin availability (10Chen J.J. London I.M. Trends Biochem. Sci. 1995; 20: 105-108Abstract Full Text PDF PubMed Scopus (272) Google Scholar). The third eIF-2 kinase, GCN2, functions in the general amino acid control pathway of yeastSaccharomyces cerevisiae. In response to starvation for any one of several different amino acids, GCN2 phosphorylation of eIF-2 stimulates the translation of GCN4 (2Wek R.C. Trends Biochem. Sci. 1994; 19: 491-496Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 4Hinnebusch A.G. J. Biol. Chem. 1997; 272: 21661-21664Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 11Dever T.E. Feng L. Wek R.C. Cigan A.M. Donahue T.F. Hinnebusch A.G. Cell. 1992; 68: 585-596Abstract Full Text PDF PubMed Scopus (609) Google Scholar, 12Wek S.A. Zhu S. Wek R.C. Mol. Cell. Biol. 1995; 15: 4497-4506Crossref PubMed Google Scholar). The GCN4 protein is a transcriptional activator of more than 30 genes involved in amino acid biosynthesis. This report centers on the regulation of the GCN2 protein kinase. The kinase catalytic domain of GCN2 shares sequence and structural similarities with the PKR and HRI that are distinguishable from other eukaryotic protein kinases (2Wek R.C. Trends Biochem. Sci. 1994; 19: 491-496Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 13Chen J.J. Throop M.S. Gehrke L. Kuo I. Pal J.K. Brodsky M. London I.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7729-7733Crossref PubMed Scopus (174) Google Scholar, 14Hinnebusch A.G. Wek R.C. Dever T.E. Cigan A.M. Feng F. Donahue T.F. Ilan J. Translation Regulation of Gene Expression. 2. Plenum Press, New York1993: 87-115Google Scholar). Adjacent to the kinase catalytic domain, GCN2 contains a region homologous to histidyl-tRNA synthetase (HisRS) that binds uncharged tRNA (12Wek S.A. Zhu S. Wek R.C. Mol. Cell. Biol. 1995; 15: 4497-4506Crossref PubMed Google Scholar, 15Wek R.C. Jackson B.M. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4579-4583Crossref PubMed Scopus (219) Google Scholar). It is proposed that different uncharged tRNAs, which accumulate during amino acid starvation conditions, can interact with the synthetase-related domain of GCN2, resulting in activation of the kinase and phosphorylation of eIF-2 (2Wek R.C. Trends Biochem. Sci. 1994; 19: 491-496Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 4Hinnebusch A.G. J. Biol. Chem. 1997; 272: 21661-21664Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, 12Wek S.A. Zhu S. Wek R.C. Mol. Cell. Biol. 1995; 15: 4497-4506Crossref PubMed Google Scholar, 15Wek R.C. Jackson B.M. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4579-4583Crossref PubMed Scopus (219) Google Scholar, 16Zhu S. Sobolev A.Y. Wek R.C. J. Biol. Chem. 1996; 271: 24989-24994Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Another domain that is important for regulation of GCN2 involves targeting of the kinase to ribosomes. Ramirez et al. (17Ramirez M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (133) Google Scholar) showed by several criteria that GCN2 was associated with ribosomes. GCN2 co-migrated with free 40 S and 60 S ribosomal subunits, 80 S particles, and polysomes separated by sucrose gradient centrifugation. When ribosomes were dissociated into 40 S and 60 S subunits by omitting MgCl2 from the extract preparation, GCN2 remained associated with 60 S ribosomal subunits (17Ramirez M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (133) Google Scholar). GCN2 was also complexed with ribosomal subunits after electrophoresis in a composite agarose-acrylamide gel under nondenaturing conditions. The related eIF-2 kinase, PKR, was also found to interact with the ribosomal machinery based on biochemical fractionation (3Samuel C.E. J. Biol. Chem. 1993; 268: 7603-7606Abstract Full Text PDF PubMed Google Scholar, 18Samuel C.E. Knutson G.S. Berry M.J. Atwater J.A. Lasky S.R. Methods Enzymol. 1986; 119: 499-516Crossref PubMed Scopus (31) Google Scholar, 19Langland J.O. Jacobs B.L. J. Biol. Chem. 1992; 267: 10729-10736Abstract Full Text PDF PubMed Google Scholar, 20Zhu S. Romano P.R. Wek R.C. J. Biol. Chem. 1997; 272: 14434-14441Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and immunofluorescent staining (21Schwemmle M. Clemens M. Hilse K. Pfeifer K. Troster H. Muller W.E.G. Bachmann M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10292-10296Crossref PubMed Scopus (69) Google Scholar). Two regions in the amino terminus of PKR, designated dsRNA-binding domains (DRBDs), contain several basic amino acids in a predicted α-helical structure that are related to a family of RNA-binding proteins (22St Johnston D. Brown N.H. Gall J.G. Jantsch M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus Google Scholar, M. S. A.G. M. Johnston D. J. 1995; PubMed Scopus Google Scholar, A. M.J. M. A. J. 1995; PubMed Scopus Google Scholar, C.E. 1992; PubMed Scopus Google Scholar). In to regulating kinase by the DRBD sequences PKR association with ribosomes S. Romano P.R. Wek R.C. J. Biol. Chem. 1997; 272: 14434-14441Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). PKR targeting to ribosomes is proposed to in phosphorylation of eIF-2 by providing the kinase access to this In this report, we the that GCN2 residues 1467–1590 can bind independently to ribosomes. This GCN2 domain contains a sequence with to the core of the of these conserved lysine residues GCN2 interaction with ribosomes and of in response to starvation for amino for the the lysine residues are for to in vitro, suggesting that interaction with rRNA mediates ribosome targeting. This cellular localization GCN2 access to eIF-2 or to regulatory ligands. these indicate that related RNA-binding sequences the in of GCN2 and PKR in response to cellular R.C. Jackson B.M. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4579-4583Crossref PubMed Scopus (219) Google Scholar) and T.E. Feng L. Wek R.C. Cigan A.M. Donahue T.F. Hinnebusch A.G. Cell. 1992; 68: 585-596Abstract Full Text PDF PubMed Scopus (609) Google Scholar) were with different of GCN2 in the GCN2 in R.C. Ramirez M. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. PubMed Scopus Google in (12Wek S.A. Zhu S. Wek R.C. Mol. Cell. Biol. 1995; 15: 4497-4506Crossref PubMed Google GCN2 in M. H. Mol. Cell. Biol. PubMed Scopus Google and R.C. Jackson B.M. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4579-4583Crossref PubMed Scopus (219) Google Scholar) is a of containing a site into GCN2 after the codon for were in with or R.C. Ramirez M. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. PubMed Scopus Google Scholar) contains into the (12Wek S.A. Zhu S. Wek R.C. Mol. Cell. Biol. 1995; 15: 4497-4506Crossref PubMed Google Scholar) and R.C. Ramirez M. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. PubMed Scopus Google Scholar) are of containing and the three lysine residues in the terminus of GCN2, at and were by to and and was into to the and into to the of GCN2 we a to from into a The resulting a sequence with GCN2 residues 1467–1590 from the is a that the this of GCN2 in Zhu S. Sobolev A.Y. Wek R.C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) or was used to the into A.M. Gene 1986; PubMed Scopus Google Scholar). The resulting the truncated GCN2 sequences from the expressed The kinase domain of GCN2 from residues to were expressed in as Zhu S. Sobolev A.Y. Wek R.C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). was as Hinnebusch A.G. Mol. Cell. Biol. PubMed Scopus Google Scholar). conditions, were in the S. A. Methods in Cold Spring Harbor Laboratory Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Scholar) with amino acids and were after for at 30 starvation conditions, were for under conditions, was to the and the was for an at 30 are the from three were expressed as hydrolyzed of were as R.C. Ramirez M. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. PubMed Scopus Google Scholar). GCN2 and were a protein R.C. Jackson B.M. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4579-4583Crossref PubMed Scopus (219) Google Scholar) and A. The was by the terminus of GCN2 and The with a of was also the in and was in containing the of GCN2 proteins were by from of different a GCN2 kinase were as with the in the of and (17Ramirez M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (133) Google Scholar, R.C. Ramirez M. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. PubMed Scopus Google Scholar). was with or were in GCN2 ribosome association the kinase was expressed from or (17Ramirez M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (133) Google Scholar). The protein was expressed containing and was expressed containing Zhu S. Sobolev A.Y. Wek R.C. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). were under and was to the were by and with and were in in the of and and containing were a sucrose gradient in as (17Ramirez M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (133) Google Scholar) and was a at for were an at and were gradient of the was also and a and in the or of as (17Ramirez M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (133) Google Scholar). containing with or was at 30 in with and was to the and for an The cell was by and with a of and were in A and with and a were by at and was a containing that binds to the of the the with A containing proteins were with in A. The of the protein was in with that predicted from the This protein was from an extract from with Additionally, GCN2 and recombinant proteins were by the terminus of GCN2. of the recombinant to we a to that by et al. J. Mathews M.B. Cell. 1986; Full Text PDF PubMed Scopus Google Scholar). of GCN2 or recombinant protein were with bound to or in a of and the for 30 at the were by at were three in and recombinant protein bound to the were by by for of were by gel electrophoresis in a gel and the recombinant protein was by staining the gel with were at a were in were and the were with a from to at a of and with an of The were structure were the of et al. Methods Enzymol. 1986; PubMed Scopus Google Scholar) and the GCN2 interaction with ribosomes is proposed to of GCN4 translation in response to amino acid association appears to the of GCN2 of this region interaction of the kinase with ribosomes (17Ramirez M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (133) Google Scholar). the terminus of GCN2 functions independently as a domain, we expressed a protein containing only GCN2 residues 1467–1590 in were as under and and by sucrose gradient The of the in gradient fraction was by the terminus of GCN2 The truncated GCN2 protein with with of found in gradient containing 60 S and 80 S and results were found the truncated GCN2 protein was expressed in that the ribosomal association was of GCN2. the GCN2 kinase catalytic sequences from to expressed in free of ribosomes in a sucrose gradient the interaction of the of GCN2 with we in the of leading to the of ribosomes into free 40 S and 60 S for the GCN2, we found that of the protein with free 60 S subunits When these were with the protein was dissociated from the 60 S This in the of was for GCN2 (17Ramirez M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (133) Google Scholar), that GCN2 is an ribosomal These results indicate that the amino acid residues from 1467 to 1590 target GCN2 kinase to ribosomes. that the DRBD sequences of PKR association of this mammalian eIF-2 kinase with ribosomes S. Romano P.R. Wek R.C. J. Biol. Chem. 1997; 272: 14434-14441Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), we are sequence similarities GCN2 residues 1467–1590 and the RNA-binding regions of Interestingly, residues to in GCN2 sequence to the core of the DRBD sequences found in PKR and other of this RNA-binding family the structure of the RNA-binding regions in PKR have been the of DRBD sequences from protein in and from that this core region is α-helical M. S. A.G. M. Johnston D. J. 1995; PubMed Scopus Google Scholar, A. M.J. M. A. J. 1995; PubMed Scopus Google Scholar). The conserved lysine residues are at the of the and are proposed to C.E. 1992; PubMed Scopus Google Scholar, S.R. Mathews M.B. 1992; PubMed Scopus Google Scholar). of the GCN2 sequence also to a of residues in an α-helical structure the of this sequence of GCN2 in the of general we the three conserved lysine residues as in 2. The resulting was into the on a or The of was in the GCN2 in the of an inhibitor of than these were under or In the of was in in response to The also showed in during amino acid conditions, the was expressed from a with the that the protein is impaired for of the general control also to on with and showed in the of a by during starvation conditions. This of the general amino acid control pathway is to of the as an of protein to be to GCN2 In the protein on the was with the was of the general control during amino acid sequences in GCN2 are for of and in response to starvation for amino of on in a in the conserved lysine residues in the region of GCN2 the ability of the kinase to stimulate GCN4 translation in response to this sequence is for GCN2 association with we cellular from or other of GCN2 and the by sucrose In an Ramirez et al. (17Ramirez M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (133) Google Scholar) GCN2 protein in gradient fraction by GCN2 and for by of the in the of This kinase which the GCN2 kinase domain was more than and in was found to be an of protein with this we found GCN2 kinase associated with free 40 60 and 80 S and a of GCN2 in the sucrose gradient we a cellular extract from the kinase from a and GCN2 protein by the protein was for ribosomal a was by sucrose gradient by the kinase that the of the protein in the kinase was that of GCN2 The protein was found the of the sucrose in free of ribosomes. In a from the kinase from a was by sucrose gradient by protein were in the cell we found the kinase in the of the gradient in containing ribosomes Two of GCN2 were to the function of other domains of the kinase were for targeting to the translation machinery. we from the protein that contains a of the conserved lysine in the sequence in the kinase catalytic domain, impaired R.C. Ramirez M. Jackson B.M. Hinnebusch A.G. Mol. Cell. Biol. PubMed Scopus Google Scholar). of the protein in the gradient a to that for GCN2 we a GCN2 protein containing in the conserved and at and in the sequence of the synthetase-related domain. this protein was to be in ability to stimulate GCN4 in response to amino acid and was for to uncharged tRNA with GCN2 (12Wek S.A. Zhu S. Wek R.C. Mol. Cell. Biol. 1995; 15: 4497-4506Crossref PubMed Google Scholar). of the protein in the sucrose gradient a ribosomal for the protein to that for the GCN2 with the ribosomal association of the kinase we that of uncharged tRNA to the domain or activation of the kinase catalytic is a for ribosomal association of GCN2. the well characterized of the DRBD regions of PKR for we to the domain of GCN2 this in a recombinant protein containing the GCN2 sequence from residues 1467 to 1590 to an sequence containing was used to the recombinant and in we and a protein containing the proteins were to as by staining of an gel after GCN2 or recombinant protein were in a containing bound to or to the were by a and of proteins bound to were by and by staining with protein containing the domain from GCN2 was found to bind association was found with The recombinant protein for that the ribosomal targeting domain of GCN2 can bind more the structure of the region of GCN2, we the of the recombinant GCN2 protein The contains a of with and and at This is of proteins and of the structure a α-helical and The recombinant protein showed a suggesting that these the structure of the GCN2 the to the we a site the and GCN2 sequences to the from the recombinant of the version of the recombinant the was to be to that in that the to the of GCN2 is a protein kinase that translation initiation in response to amino acid In this report, we that the terminus of GCN2 from residues 1467 to 1590 targeting of the kinase to the translation machinery. Interestingly, of this region of GCN2 with DRBD sequences that ribosome interaction of a related eIF-2 kinase, PKR, a sequence in GCN2 with to the core of this RNA-binding domain. Substitutions of lysine residues conserved among block association of GCN2 with ribosomes and impaired the ability of the kinase to stimulate the general control pathway in response to amino acid limitation. These results that appropriate localization of GCN2 to the translational machinery is an obligate step in the mechanism leading to kinase phosphorylation of eIF-2. of GCN2 were for their on association of the kinase to ribosomes. the containing in the was associated with the protein showed a of ribosome interaction to GCN2 GCN2 interaction with uncharged tRNA also to be a step leading to ribosome the containing in the domain that of uncharged tRNA in (12Wek S.A. Zhu S. Wek R.C. Mol. Cell. Biol. 1995; 15: 4497-4506Crossref PubMed Google Scholar), with ribosomes in the sucrose gradient These results that the sequence of GCN2 with ribosomes of of GCN2 or of kinase by uncharged tRNA that during amino acid starvation conditions. association with ribosomes in the leading to GCN2 phosphorylation of Targeting to ribosomes GCN2 access to eIF-2. During initiation of eIF-2 is associated with ribosomal subunits, providing GCN2 to (5Merrick W.C. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-70Google Scholar, M. Wek R.C. Hinnebusch A.G. Mol. Cell. Biol. 1991; 11: 3027-3036Crossref PubMed Scopus (133) Google Scholar). A of ribosome association in the regulation of GCN2 kinase is that a for GCN2 to the of uncharged tRNA in the GCN2 interaction with ribosomes be to the with the domain uncharged tRNA that and is from this site during the of step in protein synthesis. The of GCN2 in the ribosome be to that of the protein In this the synthetase of is thought to be by uncharged tRNA that binds the A site during amino acid starvation M. and and for D. Scholar). This that the uncharged tRNA in the cell can be more by GCN2 the kinase is associated with ribosomes with the kinase the In of the that a ribosomal of uncharged tRNA and Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus Google Scholar, R. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, R. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) proposed that is cellular for and of tRNA to the translation During this are from synthetases to the to the ribosomes soluble in the during the translation process, with their tRNA synthetases to the during of amino acid uncharged are and are from the A site with by this The ribosome localization of GCN2 the kinase access to one of the the synthetase-related domain of GCN2 to the of uncharged as and M.J. D. Hinnebusch A.G. Mol. Cell. Biol. 1993; PubMed Scopus (97) Google Scholar, M.J. Hinnebusch A.G. J. 1995; PubMed Scopus Google Scholar), that a associated with ribosomes and are for of GCN2 phosphorylation of eIF-2 during function to uncharged from the A site to the domain of GCN2 M.J. K. Hinnebusch A.G. Mol. Cell. Biol. 1997; PubMed Google Scholar). different proteins have been that contain DRBD sequences A. M.J. M. A. J. 1995; PubMed Scopus Google Scholar). of these proteins have been characterized only by and the of in their functions are have that sequences association of the eIF-2 GCN2 and PKR, with ribosomes. another that is the of was also found to associate with ribosomes Jantsch J. Cell. Biol. 1997; PubMed Scopus Google Scholar). Another is the protein by from S. L. PubMed Scopus Google Scholar, L. M. J. Biochem. 1992; PubMed Scopus Google Scholar) that contains a DRBD and is associated with the ribosomal subunit in The ribosomal that can by different DRBD sequences to be with ribosomal that GCN2 and PKR are to 60 S and 40 S ribosomal subunits, These different ribosomal that DRBD sequences bind to double-stranded regions in acid the be to this for different sequences and Additionally, the that proteins contain DRBD sequences that RNA-binding to the for ribosomal and for their on this for with the and and Hinnebusch for GCN2