ライフサイエンスコーパス: Met-tRNA

1 bound to the 40S preinitiation complex (40S.Met-tRNA(i).eIF2.GTP), promoted by eIF5, must occur only

2 nhibits the eIF3-dependent conversion of 40S/Met-tRNA(i)(Met)/mRNA to translationally competent 80S/M

3 )(Met)/mRNA to translationally competent 80S/Met-tRNA(i)(Met)/mRNA initiation complexes to repress co

4 r complex containing eIFs 1, 2, 3, and 5 and Met-tRNA(i)(Met), whose formation is required for an opt

5 tifactor complex (MFC) with eIFs 1, 2, 5 and Met-tRNA(i)(Met).

6 ation with eIF3 promotes binding of eIF2 and Met-tRNA(i)(Met) to 40S ribosomes.

7 y long-lived ribosome complex with eIF5B and Met-tRNA(i)(Met) immediately before transition into elon

8 ernary complex (TC) comprising eIF2, GTP and Met-tRNA(i).

9 at facilitates ribosomal subunit joining and Met-tRNA(i) binding to ribosomes in all three kingdoms o

10 of eIF2gamma that bind the alpha-subunit and Met-tRNA(i)(Met), respectively.

11 Gly-tRNA, Ala-tRNA, Ser-tRNA, Pro-tRNA, and Met-tRNA.

12 n of acceptor proteins using Leu-, Phe-, and Met-tRNAs as amino acid donors.

13 g in vitro binding of initiator Met-tRNA (as Met-tRNA(i).eIF2.GTP ternary complex) to 40 S ribosomal

14 ith the 40S initiation complex (40S*eIF3*AUG*Met-tRNA(f)*eIF2*GTP) and, acting as a GTPase activating

15 ith the 40S initiation complex (40S-eIF3-AUG-Met-tRNA(f)-eIF2-GTP) to promote the hydrolysis of ribos

16 S ribosomal initiation complex (40S.eIF3.AUG.Met-tRNA(f).eIF2.GTP) to promote the hydrolysis of bound

17 pendent of the canonical initiator tRNA (AUG/Met-tRNA(i)(Met)) pathway but required expression of euk

18 Met) ternary complex on base-pairing between Met-tRNA(i)(Met) and the start codon.

19 and eIF2 in eukaryotes are GTPases that bind Met-tRNA(i)(Met) to the small ribosomal subunit.

20 Translation initiation factor 2 (eIF2) binds Met-tRNA(i)(Met) to the 40S ribosomal subunit, and previ

21 In vitro, the MFC-GTP binds Met-tRNA(i) and delivers the tRNA to the ribosome at the

22 , a translation initiation factor that binds Met-tRNA(i), tryptic peptides from rabbit reticulocyte e

23 1:1 stoichiometry with respect to the bound Met-tRNA(i).

24 ynthetase (MRS) and production of a chimeric Met-tRNA(Ile) that would compromise translational fideli

25 IF3/eIF1 in a multifactor complex containing Met-tRNA(i)(Met).

26 F2 is a heterotrimer that binds and delivers Met-tRNA(i)(Met) to the 40 S ribosomal subunit in a GTP-

27 terotrimeric complex that binds and delivers Met-tRNA(i)(Met) to the 40 S ribosomal subunit in a GTP-

28 terial ribosomes, are critical for efficient Met-tRNA(i)(Met) binding and AUG selection in eukaryotes

29 In cooperation with eIF3, eIF1, and eIF1A, Met-tRNA(Met)(i)/eIF2/GTP binds to 40S subunits yielding

30 preinitiation complex (40 S.eIF3.eIF1.eIF1A.Met-tRNA(i).eIF2.GTP).

31 itiation factor (eIF) 3 and the ternary eIF2/Met-tRNA(i)(Met)/GTP complex and subsequently domain II

32 n eukaryotic translation initiation, eIF2GTP-Met-tRNA(i)(Met) ternary complex (TC) interacts with eIF

33 S preinitiation complex (40S.eIF1.eIF1A.eIF3.Met-tRNA(i).eIF2.GTP) and the subsequent binding of the

34 tRNA genes, the 5S rRNA gene (rrf), and an f-Met tRNA gene.

35 m of rrs and immediately downstream of the f-Met tRNA gene.

36 initiation was reduced with the fluorophore-Met-tRNA(f) compared with fMet-tRNA(f) with pyrene havin

37 s systems that can separate fMet-tRNA(fMet), Met-tRNA(fMet), and tRNA(fMet) shows that there is no fo

38 version of GTP to GDP on eIF2's affinity for Met-tRNA(i) in solution.

39 at-treated prt1-1 extracts are defective for Met-tRNA(i)Met binding to 40S subunits, and we also obse

40 Thus, three distinct pathways for Met-tRNA(i) delivery to the 40S ribosomal subunit are id

41 d in cell lysates, it may be responsible for Met-tRNA(i)-40S ribosome binding in vivo, possibly toget

42 romoting the GTP-dependent binding of formyl-Met-tRNA to the small subunit of either E. coli or bovin

43 spiratory defect in cells lacking formylated Met-tRNA(f)(Met) due to loss of the MIS1 gene that encod

44 factor might be unnecessary when formylated Met-tRNA(f)(Met) is present but becomes essential when o

45 requiring hydrolysis of GTP in the eIF2*GTP*Met-tRNA(i)(Met) ternary complex (TC) and subsequent P(i

46 lerate the rate of ternary complex (eIF2*GTP*Met-tRNA(i)(Met)) binding to 40S but only eIF1A stabiliz

47 en implicated in recruitment of the eIF2-GTP-Met-tRNA i Met ternary complex (TC) and ribosomal scanni

48 , which stimulates formation of the eIF2-GTP-Met-tRNA(i)(Met) ternary complex (TC) in a manner inhibi

49 nd indirectly through eIF5 with the eIF2-GTP-Met-tRNA(i)(Met) ternary complex (TC) to form the multif

50 of eIF1A impairs recruitment of the eIF2-GTP-Met-tRNA(i)(Met) ternary complex to 40S subunits, elimin

51 ormation and decreases the level of eIF2-GTP-Met-tRNA(i)(Met) ternary complexes.

52 multifactor complex (eIF1-eIF3-eIF5-eIF2-GTP-Met-tRNA(i)(Met)).

53 ation complexes containing eIF3 and eIF2-GTP-Met-tRNA(iMet) to bind directly to the initiation codon,

54 e-initiator methionyl transfer RNA (eIF2.GTP.Met-tRNA(i )(Met)).

55 bly of the Saccharomyces cerevisiae eIF2.GTP.Met-tRNA(i) ternary complex and have determined the effe

56 GCN4 translation due to limiting of eIF2.GTP.Met-tRNA(i)(Met) ternary complex binding to the ribosome

57 primarily to regulate the level of eIF2.GTP.Met-tRNA(i)(Met) ternary complexes in vivo.

58 he eukaryotic initiation factor 2 (eIF2)/GTP/Met-tRNA(iMet) ternary complex (TC) and promotes scannin

59 aryotic translation initiation, the eIF2.GTP/Met-tRNA(i)(Met) ternary complex (TC) binds the eIF3/eIF

60 hroughout initiation: it stimulates eIF2/GTP/Met-tRNA(i)(Met) attachment to 40S ribosomal subunits, s

61 First, eIF5 binds eIF2/GTP/Met-tRNA(i)(Met) ternary complex (TC), promoting its rec

62 F5 stimulates GTP hydrolysis by the eIF2/GTP/Met-tRNA(i)(Met) ternary complex on base-pairing between

63 c initiation factor (eIF) 3 and the eIF2/GTP/Met-tRNA(i)(Met) ternary complex.

64 t interact with eIF1, eIF5, and the eIF2/GTP/Met-tRNA(i)(Met) ternary complex.

65 th enhance ribosomal recruitment of eIF2/GTP/Met-tRNA(i)(Met), but have opposite effects on the strin

66 irectly implicating eIF5-CTD in the eIF2/GTP/Met-tRNA(i)Met ternary complex binding process required

67 canonical UUG codon, presumably by impairing Met-tRNA(i)(Met) binding.

68 ma-N135D GTP-binding domain mutation impairs Met-tRNA(i)(Met) binding and causes a Sui(-) phenotype.

69 An eIF2gamma-A219T mutation impairs Met-tRNA(i)(Met) binding but unexpectedly enhances the f

70 also can sense and bind uncharged initiator Met tRNA, resulting in the sequestering of the anti-Shin

71 to some extent utilize formylated initiator Met-tRNA to initiate protein synthesis and that initiati

72 ation in vivo of the mitochondrial initiator Met-tRNA in these strains.

73 eas a formylation-defective mutant initiator Met-tRNA, which binds to MTF with approximately the same

74 complex using in vitro binding of initiator Met-tRNA (as Met-tRNA(i).eIF2.GTP ternary complex) to 40

75 ng 80S ribosomes in the absence of initiator Met-tRNA(i) or any canonical initiation factors, from a

76 teraction between the anticodon of initiator Met-tRNA, associated with eIF2-GTP and 40S ribosomal sub

77 a tRNA-like IRES directs precise, initiator Met-tRNA-independent translation of two overlapping ORFs

78 t formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the pur

79 We show that the substrate initiator Met-tRNA protects MTF against trypsin cleavage, whereas

80 stem and the 3'-end region of the initiator Met-tRNA against cleavage by double and single strand-sp

81 In contrast, the initiator Met-tRNA is formylated in the respective "wild-type" par

82 eIF2 is a GTPase that delivers the initiator Met-tRNA to the P site on the small ribosomal subunit du

83 anticodon interactions between the initiator Met-tRNA(i)(Met) and the mRNA.

84 in the correct positioning of the initiator Met-tRNA(i)(Met) on the ribosome in the later stages of

85 stly with the acceptor stem of the initiator Met-tRNA, which contains the critical determinants for f

86 mylation) are not protected by the initiator Met-tRNA.

87 rus can form 80S ribosomes without initiator Met-tRNA, eIF2, or GTP hydrolysis, with a CCU triplet in

88 lular eIF2 forms a complex with eIF5 lacking Met-tRNA(i)(Met), and here we investigate its physiologi

89 d both by eIF2/eIF3- and eIF5B/eIF3-mediated Met-tRNA(iMet) recruitment were destabilized by eIF1, di

90 Since an MFC-Met-tRNA(i) complex is detected in cell lysates, it may

91 portance of the formylation of mitochondrial Met-tRNA for the interaction with IF-2(mt) was investiga

92 icates that the formylation of mitochondrial Met-tRNA specifies its participation in initiation throu

93 will not effectively stimulate mitochondrial Met-tRNA binding to mitochondrial ribosomes, exhibiting

94 lated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initi

95 dergoes an induced fit in the functional MTF.Met-tRNA complex but not in the nonfunctional one.

96 lso depends on formation of a functional MTF.Met-tRNA complex.

97 s cerevisiae can initiate with nonformylated Met-tRNA(f)(Met), as demonstrated in yeast mutants in wh

98 We could rescue 40S binding of Met- tRNA(i)Met and mRNA, and translation of luciferase

99 conformation to enable full accommodation of Met-tRNA(i)(Met) in the P site for AUG selection.

100 to the evolution of splicing.The binding of Met-tRNA to ribosomes is mediated by a GTP-binding prote

101 ut in vitro formylation increases binding of Met-tRNA(f)(Met) to translation initiation factor 2 (IF2

102 ctor complex in vivo, reduced the binding of Met-tRNA(i)(Met) and mRNA to 40S subunits in vitro.

103 propose that eIF5-CTD stimulates binding of Met-tRNA(i)(Met) and mRNA to 40S subunits through intera

104 n eIF2gamma subtly alter the conformation of Met-tRNA(i)(Met) on the 40S subunit and thereby affect t

105 The delivery of Met-tRNA(i) to the 40S ribosomal subunit is thought to o

106 iol and activated methionine, in the form of Met-tRNA, are incubated with MetRS.

107 ly in purine biosynthesis and formylation of Met-tRNA and indirectly in the biosynthesis of methionin

108 ty of start codon recognition independent of Met-tRNA(i)(Met) binding affinity.

109 A portion of Met-tRNA(Met) is formylated for initiation, whereas the

110 d SE2, was found to stimulate recruitment of Met-tRNA(i)(Met) in the ternary complex (TC) with eIF2.G

111 te efficient eIF2-independent recruitment of Met-tRNA(Met)(i) to 40S/mRNA complexes, if attachment of

112 ve interaction with the methionine moiety on Met-tRNA(i) that is disrupted when GTP is replaced with

113 , the NTT does not clash with either mRNA or Met-tRNA(i)(Met), consistent with its suggested role in

114 antly alter binding of guanine nucleotide or Met-tRNA(i)(Met) ligands by eIF2 in vitro.

115 ting a 50-fold preference for fMet-tRNA over Met-tRNA in this assay.

116 ic of mobile elements, including a potential Met-tRNA priming site, similar to that found in retrotra

117 report that eIF5B or eIF5B/eIF3 also promote Met-tRNA(iMet) binding to IRES-40S complexes, forming 48

118 ses two functions: synthetic, which provides Met-tRNA for protein synthesis, and editing, which rejec

119 n by eIF2 phosphorylation, despite requiring Met-tRNA(Met)(i).

120 A208V and A382V suppressor mutations restore Met-tRNA(i)(Met) binding affinity and cell growth; howev

121 f the ribosome, with initiator transfer RNA (Met-tRNA(i)(Met)) positioned over the start codon of mes

122 osin, pyrene, or coumarin attached to [(35)S]Met-tRNA(f).

123 anslational defect suggests eIF5B stabilizes Met-tRNA(i)(Met) binding and that GTP hydrolysis by eIF5

124 C) with eIF1, eIF2, and eIF5 that stimulates Met-tRNA(i)(Met) binding to 40S ribosomes and promotes s

125 er affinity for mitochondrial fMet-tRNA than Met-tRNA, using either the native mitochondrial tRNA(Met

126 40S ribosomes, creating the possibility that Met-tRNA(i) might bind directly to such 40S-factor compl

127 alysis to study the interactions between the Met-tRNA(fMet) and MTF in solution.

128 vitro or on the puromycin reactivity of the Met-tRNA in the 80S initiation complex.

129 MFC-GDP shows a greatly reduced affinity to Met-tRNA(i) compared to that for eIF2-GDP, suggesting th

130 mulates the GTPase activity of eIF2 bound to Met-tRNA(i)(Met), and its C-terminal domain (eIF5-CTD) b

131 nctions in binding methionyl initiator tRNA (Met-tRNA(i)(Met)) to the ribosome and in selecting AUG c

132 te it delivers the methionyl initiator tRNA (Met-tRNA(i)) to the small ribosomal subunit and releases

133 recruitment of aminoacylated initiator tRNA (Met-tRNA(Met)(i)) by eukaryotic initiation factor eIF2.

134 formylation of the initiator methionyl-tRNA (Met-tRNA(fMet)).

135 ect binding of the initiator methionyl-tRNA (Met-tRNA(i)) to 40 S ribosomal subunits in a codon-depen

136 N-Formylation of initiator methionyl-tRNA (Met-tRNA(Met)) by methionyl-tRNA formyltransferase (MTF)

137 fic formylation of initiator methionyl-tRNA (Met-tRNA) by methionyl-tRNA formyltransferase (MTF) is i

138 E. coli enzyme and initiator methionyl-tRNA (Met-tRNA) by using two complementary protection experime

139 ctor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation ini

140 Compared to human tRNA gene promoters (tRNA(Met), tRNA(Val)), the human small nuclear RNA U6 gene (U

141 as the primer, can be forced to utilize tRNA(Met), tRNA(1,2)(Lys), tRNA(His), or tRNA(Glu), although

142 ons on the stability of methionylated tRNAi (Met-tRNA(i)) binding (in the ternary complex [TC] with e

143 The NTT and CTT both thread under Met-tRNA(i)(Met) reaching into the P-site.

144 ondrial IF2 (mIF2) in utilizing unformylated Met-tRNA(f)(Met).

145 ith eIF2Bdelta leading to a competition with Met-tRNA(i).

146 e 40 S subunit in a multifactor complex with Met-tRNA(i)(Met), eIF2, eIF3, and eIF5 and binds near th

147 bility of eIF3 to stabilize the eIF2 x GTP x Met-tRNA(i) ternary complex.