ライフサイエンスコーパス: eIF5B

1 eIF5B did not influence factor release in the absence of

2 eIF5B has also been shown to promote the translation of

3 eIF5B promotes 60S ribosome subunit joining and pre-40S

4 eIF5B, the eukaryotic ortholog of IF2, is a GTPase that

5 on initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to di

6 al tail (eIF1A-CTT) binds to eIF5B Domain-4 (eIF5B-D4).

7 eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal

8 eukaryotic translation initiation factor 5B (eIF5B) from Saccharomyces cerevisiae impaired cell growt

9 otein named eukaryotic initiation factor 5B (eIF5B) in eukaryotes.

10 Eukaryotic initiation factor 5B (eIF5B) is a GTPase that facilitates joining of the 60 S

11 nce time of eukaryotic initiation factor 5B (eIF5B) on the 80S ribosome after the joining of individu

12 initiation, eukaryotic initiation factor 5B (eIF5B) promotes the 60S subunit joining with the 40S ini

13 eukaryotic translation initiation factor 5B (eIF5B), the eukaryotic ortholog of bacterial initiation

14 ction with translation initiation factor 5B (eIF5B).

15 eukaryotic translation initiation factor 5B (eIF5B).

16 wo protein factors, IF1 (a/eIF1A) and IF2 (a/eIF5B), are conserved among all three kingdoms of life a

17 reestablish coupling between GTP binding and eIF5B domain movements.

18 cate that the interactions between eIF1A and eIF5B are being continuously rearranged during translati

19 rved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexe

20 cular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.

21 The factors eIF1A and eIF5B from eukaryotes show extensive amino acid sequence

22 e binding between the C termini of eIF1A and eIF5B has implications for eukaryote-specific mechanisms

23 In contrast, yeast eIF1A and eIF5B have been reported to interact in the absence of r

24 tural model for the interaction of eIF1A and eIF5B in the context of the ribosome is presented.

25 intramolecular interactions within eIF1A and eIF5B interfere with one or both eIF5B/eIF1A contact int

26 e evolutionarily conserved factors eIF1A and eIF5B plays an important role in translation initiation,

27 Mutational analysis of eIF1A and eIF5B revealed distinct functions of eIF5B in 48S IC for

28 s is more complex and accordingly, eIF1A and eIF5B seem to have acquired a number of new functions wh

29 We propose that eIF1A and eIF5B simultaneously interact at two sites that are >50

30 tion complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy.

31 ation, and in vitro binding assays eIF1A and eIF5B were found to interact directly, and the eIF1A bin

32 ic homologs, eukaryotic IFs (eIFs) eIF1A and eIF5B, has only recently become evident.

33 s require hydrolysis of GTP by both eIF2 and eIF5B to complete translation initiation.

34 A knockout of both eIF2A and eIF5B yielded a "synthetically sick" yeast strain with a

35 of 48S complexes assembled by eIF2/eIF3- and eIF5B/eIF3-mediated mechanisms to eIF1-induced destabili

36 S complexes assembled both by eIF2/eIF3- and eIF5B/eIF3-mediated Met-tRNA(iMet) recruitment were dest

37 Alternative model that eIF5 and eIF5B cause 43S pre-initiation complex rearrangement fav

38 Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation-co

39 Here we show that eIF5 and eIF5B together stimulate 48S IC formation influencing in

40 c suppressors restored yeast cell growth and eIF5B nucleotide-binding, GTP hydrolysis, and subunit jo

41 irs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supp

42 The uncoupling of translation and eIF5B GTPase activity suggests a regulatory rather than

43 ciation of eIF2*GDP from initiator tRNA, and eIF5B is then required to stabilize the initiator tRNA i

44 s an antagonist of G0 and G0-like states, as eIF5B depletion reduces maturation of G0-like, immature

45 U, suggesting functional interaction between eIF5B and the A loop.

46 (iii) an intramolecular interaction between eIF5B-D3 and -D4.

47 IF1A: (i) a second binding interface between eIF5B and eIF1A; (ii) a dynamic intramolecular interacti

48 rolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation

49 n eIF1A and eIF5B interfere with one or both eIF5B/eIF1A contact interfaces, but are disrupted on the

50 h case Met-tRNAiMet is recruited directly by eIF5B.

51 Following subunit joining, GTP hydrolysis by eIF5B alters the conformation of the final initiation co

52 ing 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and t

53 sent a kinetic analysis of GTP hydrolysis by eIF5B in the context of the translation initiation pathw

54 A(i)(Met) binding and that GTP hydrolysis by eIF5B is a checkpoint monitoring 80S ribosome assembly i

55 dicate that stimulation of GTP hydrolysis by eIF5B requires the completion of early steps in translat

56 Full activation of GTP hydrolysis by eIF5B requires the extreme C terminus of eIF1A, which ha

57 joining, is accelerated by GTP hydrolysis by eIF5B.

58 d dynamic fidelity checkpoint implemented by eIF5B in concert with components of the large ribosomal

59 Consistently, eIF5B overexpression promotes maturation of G0-like imma

60 man eIF5B GTP-binding domain to Asn converts eIF5B to an XTPase and introduces an XTP requirement for

61 titution assays showed that C2879U decreased eIF5B-catalyzed 60S subunit joining with a 40S IC.

62 The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo

63 Here, we determined eIF5B's position on 80S ribosomes by directed hydroxyl r

64 These findings support the idea that eIF1A-eIF5B association is instrumental in releasing eIF1A fro

65 We present a model how the dynamic eIF1A/eIF5B interaction network can promote remodeling of the

66 show that the cleavage of initiation factor eIF5B during enteroviral infection, along with the viral

67 The translation initiation factor eIF5B gates the transition from initiation (I) to elonga

68 Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that is responsible

69 Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that mediates displ

70 This step is catalyzed by initiation factor eIF5B.

71 Translation initiation factor eIF5B/IF2 is a GTPase that promotes ribosomal subunit jo

72 iated through the general translation factor eIF5B (Fun12p in S. cerevisiae).

73 on-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunit

74 gulatory rather than mechanical function for eIF5B GTP hydrolysis in translation initiation.

75 A binding domains that has been proposed for eIF5B.

76 guanine nucleotides dissociated rapidly from eIF5B (k-1mant-GTP approximately 22-28 s-1, k-1mant-GDP

77 We show that either the absence of Fun12p (eIF5B), or a defect in eIF5, proteins involved in 60S ri

78 ) follows the order: eIF4G > eIF1A > eIF4E > eIF5B.

79 pre-40S maturation also requires the GTPase eIF5B and the ATPase Rio1.

80 In eukaryotes, the GTPase eIF5B collaborates in the correct positioning of the ini

81 The GTPase eIF5B gates progression to elongation during the second

82 A second GTPase, eIF5B, catalyzes the joining of the 60S subunit to produ

83 Our results illustrate how eIF5B transmits the presence of a properly delivered ini

84 Mutation of the conserved Asp-759 in human eIF5B GTP-binding domain to Asn converts eIF5B to an XTP

85 zyme, inactive IF2/eIF5B.GDP, and active IF2/eIF5B.GTP.

86 universal translation initiation factor IF2/eIF5B have been determined in three states: free enzyme,

87 d in three states: free enzyme, inactive IF2/eIF5B.GDP, and active IF2/eIF5B.GTP.

88 The conserved core of IF2/eIF5B consists of an N-terminal G domain (I) plus an EF-

89 st five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S compl

90 structure reveals conformational changes in eIF5B, initiator tRNA, and the ribosome that provide ins

91 itical requirement for this Switch II Gly in eIF5B, intragenic suppressors were identified in the Swi

92 We identified a stretch of residues in eIF5B, located close to the gamma-phosphate of GTP and c

93 The increased eIF5B levels lead to increased eIF5B complexes with tRNA

94 The increased eIF5B levels lead to increased eIF5B complexes with tRNA-Met(i) upon serum starvation o

95 bosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognit

96 we report three novel interactions involving eIF5B and eIF1A: (i) a second binding interface between

97 Likewise, eIF5B and eIF1A remained associated with 80S complexes f

98 In the resulting model, eIF5B is located in the intersubunit cleft of the 80S ri

99 utation restore general translation, but not eIF5B GTPase activity.

100 P hydrolysis and general translation but not eIF5B subunit joining function.

101 rminus of eIF1A also disrupts the ability of eIF5B to facilitate subunit joining.

102 and ribosomal subunit joining activities of eIF5B.

103 Inhibition of the GTPase activity of eIF5B after the joining of ribosomal subunits prevented

104 or mutations reduce the ribosome affinity of eIF5B and increase AUG skipping/leaky scanning.

105 The affinity of eIF5B for mant-GTP (Kd approximately 14-18 microM) was a

106 The affinity of eIF5B for mant-GTPgammaS was about 2 times lower (Kd app

107 by lowering the ribosome binding affinity of eIF5B.

108 cleavage data also indicate that binding of eIF5B might induce conformational changes in both subuni

109 ropose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining.

110 We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumul

111 somal subunits prevented the dissociation of eIF5B from the 80S complex, thereby preventing elongatio

112 findings illustrate how the dissociation of eIF5B serves as a kinetic checkpoint for the transition

113 een human eIF1A and the C-terminal domain of eIF5B by using solution NMR.

114 F1A and eIF5B revealed distinct functions of eIF5B in 48S IC formation and subunit joining.

115 g), that contacts the catalytic histidine of eIF5B (H480).

116 Hydrolysis of eIF5B-bound GTP is not required for its function in subu

117 nit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with express

118 h I element and at a residue in domain II of eIF5B that interacts with Switch II.

119 Overexpression of eIF5B specifically suppressed the phenotype caused by C2

120 Consistently, overexpression of eIF5B suppresses the growth and translation initiation d

121 This portion of eIF5B was found to be critical for growth in vivo and fo

122 These properties of eIF5B suggest a rapid spontaneous GTP/GDP exchange on eI

123 site was mapped to the C-terminal region of eIF5B.

124 t is necessary for the subsequent release of eIF5B from assembled 80S ribosomes.

125 osome that provide insights into the role of eIF5B in translational initiation.

126 us of eIF1A interacts with the C-terminus of eIF5B, a factor that stimulates 40S-60S subunit joining,

127 een shown to interact with the C terminus of eIF5B.

128 Leveraging the long residence time of eIF5B on the ribosome recently identified by single-mole

129 gest a rapid spontaneous GTP/GDP exchange on eIF5B and are therefore consistent with it having no req

130 We report that eIF5B or eIF5B/eIF3 also promote Met-tRNA(iMet) binding to IRES-4

131 IF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining.

132 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase

133 The translational defect suggests eIF5B stabilizes Met-tRNA(i)(Met) binding and that GTP h

134 A second factor termed eIF5B (relative molecular mass 175,000) is essential for

135 We find that eIF5B is a limiting factor for translation in these thre

136 Importantly, we find that eIF5B is an antagonist of G0 and G0-like states, as eIF5

137 7 microM) than for mant-GTP, indicating that eIF5B tolerates modifications of the triphosphate moiety

138 We report that eIF5B or eIF5B/eIF3 also promote Met-tRNA(iMet) binding

139 Here we show that eIF5B GTPase activity is required for protein synthesis.

140 We show that eIF5B mutations in Switch I, an element conserved in all

141 e site, impairing nucleotide binding and the eIF5B domain movements associated with GTP binding.

142 40S subunits during, rather than before, the eIF5B-mediated subunit joining event.

143 However, 48S complexes formed by the eIF5B/eIF3-mediated mechanism on the truncated IRES coul

144 py (cryo-EM) to determine a structure of the eIF5B initiation complex to 6.6 angstrom resolution from

145 ic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering

146 Thus, eIF5B interaction with the peptidyl transferase center A

147 he kinetics of guanine nucleotide binding to eIF5B by a fluorescent stopped-flow technique using fluo

148 d eIF1A C-terminal tail (eIF1A-CTT) binds to eIF5B Domain-4 (eIF5B-D4).

149 eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit

150 at occur during the transition from eIF2- to eIF5B-containing 48S complexes and prepare them for subu

151 Our study linked the Kozak motif to eIF5B-mediated I-E transition during translation and unc

152 e resulting 80 S initiation complex triggers eIF5B to hydrolyze its bound GTP, reducing the affinity

153 st strains expressing C-terminally truncated eIF5B.

154 iate at 3.6 angstrom allowed us to visualize eIF5B in its ribosome-bound conformation.

155 e naturally long-lived ribosome complex with eIF5B and Met-tRNA(i)(Met) immediately before transition

156 interaction of the C terminus of eIF1A with eIF5B promotes ribosomal subunit joining and possibly pr

157 ning through its C-terminal interaction with eIF5B, and eIF1A release from the initiating ribosome, w

158 yeast components and designed to stall with eIF5B and a nonhydrolyzable GTP analog.