ライフサイエンスコーパス: tRNA(Gln)

1 tRNA(Gln) import into mammalian mitochondria proceeds by

2 tRNA(Gln) was mainly cytosolic in localization; tRNA(Ile

3 Northern blot analysis revealed the sup70-65 tRNA(Gln)(CUG) is unstable, inefficiently charged, and 8

4 verting the M. thermautotrophicus GluRS to a tRNA(Gln) specific enzyme, solely through the addition o

5 upon to directly examine how GluRS2 acquired tRNA(Gln) specificity.

6 ases that form the misacylated tRNA(Asn) and tRNA(Gln) species.

7 is of the crystal structure of tRNA(Cys) and tRNA(Gln) implicated long-range tertiary base-pairs abov

8 t glutamylates both apicoplast tRNA(Glu) and tRNA(Gln), determined its kinetic parameters, and demons

9 fication at U34 of tRNA(Lys), tRNA(Glu), and tRNA(Gln) causes ribosome pausing at the respective codo

10 ations at U(34) of tRNA(Lys), tRNA(Glu), and tRNA(Gln) in maintenance of mitochondrial genome, mitoch

11 ne tRNAs including tRNA(Lys), tRNA(Glu), and tRNA(Gln) in mto2/mss1, mto2/mto1, and mto2/mto1/mss1 st

12 cation at U(34) of tRNA(Lys), tRNA(Glu), and tRNA(Gln), caused by the combination of eliminating the

13 ) in mitochondrial tRNA(Lys), tRNA(Glu), and tRNA(Gln).

14 herichia coli glutaminyl-tRNA synthetase and tRNA(Gln) have been shown to determine the apparent affi

15 ne tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA).

16 , when marked versions of tRNA(Gln)(UUG) and tRNA(Gln)(UUA) flanked by identical sequences are expres

17 y of specific interactions between GlnRS and tRNAGln determines amino acid affinity.

18 that specific interactions between GlnRS and tRNAGln ensure the accurate positioning of the 3' termin

19 sequence-specific contacts between GlnRS and tRNAGln.

20 st strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was o

21 s and use either tRNAGlu or both tRNAGlu and tRNAGln.

22 sence of GatDE has favored a unique archaeal tRNA(Gln) that may be preventing the acquisition of glut

23 Because tRNA(Gln)(UUG) is a constituent of mitochondrial RNA fra

24 dimensional structure of the complex between tRNAGln and glutaminyl-tRNA synthetase shows that the en

25 A synthetase (GluRS) that aminoacylates both tRNA(Gln) and tRNA(Glu) with glutamate.

26 We also show that both tRNA(Gln) species and a bacterial pre-tRNA(Asp) can be i

27 on of m(1)G9-containing tRNAs codons read by tRNA(Gln(TTG)), tRNA(Arg(CCG)), and tRNA(Thr(CGT)) These

28 A specific allele of the SUP70/CDC65 tRNAGln gene (sup70-65) has been reported to be defectiv

29 This maintains normal charged tRNAGln levels despite Gln4p depletion, confirmed experi

30 introduction of G15-G48 into the non-cognate tRNA(Gln) tertiary core then significantly impairs CysRS

31 E. coli tRNA(Gln) possesses the canonical Pu15-Py48 trans pairin

32 Here, guided by the core of E. coli tRNA(Gln), we sought to test and identify alternative fu

33 However, the core of E. coli tRNA(Gln), which contains G15:C48, is functional for cys

34 and in E. coli, but does not charge E. coli tRNAGln.

35 noacylation determinants of Escherichia coli tRNAGln in a genetic and biochemical analysis of suppres

36 Saccharomyces cerevisiae imports cytoplasmic tRNA(Gln) into the mitochondrion without any added prote

37 e showed in vivo localization of cytoplasmic tRNAGln in mitochondria and demonstrated its role in mit

38 rthermore reconstituted in vitro cytoplasmic tRNAGln import into mitochondria by a novel mechanism.

39 complemented by overexpressing CAA-decoding tRNA(Gln)(UUG), an inefficient wobble-decoder of CAG.

40 iae, the SUP70 gene encodes the CAG-decoding tRNA(Gln)(CUG).

41 rearranged, with the suite of genes encoding tRNA(Gln), the control region, and tRNA(Ile) located dow

42 Eukaryotic tRNA(Gln) and tRNA(Glu) recognition determinants are fou

43 RS also separately differentiated to exclude tRNA(Gln) as a substrate, and the resulting discriminati

44 ear genes, and because ectopically expressed tRNA(Gln)(UUG) fractionates with mitochondria like its e

45 uces Gln4 variants with reduced affinity for tRNA(Gln), consistent with a hinge-closing mechanism pro

46 king the NTD has reduced complementarity for tRNA(Gln) and glutamine.

47 noacylate tRNA(Glu)versus those specific for tRNA(Gln) emerged and was experimentally validated.

48 ognate tRNAs but to a decreased affinity for tRNAGln.

49 NAPhe and tRNAAsp in the free state, and for tRNAGln complexed with glutaminyl-tRNA synthetase (GlnRS

50 AGlu, whereas GluRS2 was specific solely for tRNAGln.

51 A(Trp(CCA)) are substrates for Cm formation, tRNA(Gln(UUG)), tRNA(Pro(UGG)), tRNA(Pro(CGG)) and tRNA(

52 ey feature that distinguishes tRNA(Glu) from tRNA(Gln) is the third position in the anticodon of each

53 transamidation pathway operates only for Gin-tRNAGln formation in this organism, and possibly in all

54 Gln-tRNA(Gln) is synthesized from Glu-tRNA(Gln) in most micr

55 as being able to form Asn-tRNA(Asn) and Gln-tRNA(Gln), our data demonstrate that while the enzyme is

56 the synthesis of both Asn-tRNA(Asn) and Gln-tRNA(Gln).

57 GatCAB enzyme required in vivo for both Gln-tRNA(Gln) and Asn-tRNA(Asn) synthesis.

58 modynamic framework for two-step cognate Gln-tRNA(Gln) synthesis demonstrates that the misacylating a

59 elongation factor binding to the cognate Gln-tRNA(Gln) together permit accurate protein synthesis wit

60 f an indirect aminoacylation pathway for Gln-tRNA(Gln) biosynthesis in Plasmodium that we hypothesize

61 Here, we show a similar complex for Gln-tRNA(Gln) formation in Methanothermobacter thermautotrop

62 2 nM) sequesters the tRNA synthetase for Gln-tRNA(Gln) formation, with GatDE reducing the affinity of

63 GatCAB can be similarly used for Gln-tRNA(Gln) formation.

64 hetases to synthesize Asn and GatCAB for Gln-tRNA(Gln) synthesis, their AspRS enzymes were thought to

65 Many bacteria form Gln-tRNA(Gln) and Asn-tRNA(Asn) by conversion of the misacyl

66 the GatCAB amidotransferase, which forms Gln-tRNA(Gln).

67 Glutaminyl-tRNA synthetase generates Gln-tRNA(Gln) 10(7)-fold more efficiently than Glu-tRNA(Gln)

68 aminyl-tRNA synthetase (GlnRS); instead, Gln-tRNA(Gln) is produced via an indirect pathway: a glutamy

69 nto Asn-tRNA(Asn) and Glu-tRNA(Gln) into Gln-tRNA(Gln); (iv) the TonB receptors and ferric siderophor

70 ryotic enzyme, whereas in other kingdoms Gln-tRNA(Gln) is primarily synthesized by first forming Glu-

71 Measurements of Gln-tRNA(Gln) interactions at the ribosome A-site show that

72 e phylogenetically diverse mechanisms of Gln-tRNA(Gln) synthesis.

73 enzyme is rate-limiting for synthesis of Gln-tRNA(Gln).

74 e level as ATP, but without formation of Gln-tRNA(Gln).

75 alternate pathway for the production of Gln-tRNA(Gln): misacylated Glu-tRNA(Gln) is transamidated by

76 isms lacking Gln-tRNA synthetase produce Gln-tRNA(Gln) from misacylated Glu-tRNA(Gln) through the tra

77 ormation, is not stable through product (Gln-tRNA(Gln)) formation, and has no major effect on the kin

78 termediate to form the cognate products, Gln-tRNA(Gln) or Asn-tRNA(Asn).

79 estor, used transamidation to synthesize Gln-tRNA(Gln) and that both the Bacteria and the Archaea ret

80 zymes found in organisms that synthesize Gln-tRNA(Gln) by an alternative pathway.

81 These data show that Gln-tRNA(Gln) biosynthesis in the Plasmodium apicoplast proc

82 Glu-tRNA(Gln), followed by conversion to Gln-tRNA(Gln) by a tRNA-dependent amidotransferase.

83 ombinant GatAB converts Glu-tRNA(Gln) to Gln-tRNA(Gln) in vitro.

84 otransferase to convert Glu-tRNA(Gln) to Gln-tRNA(Gln) needed for protein synthesis.

85 were unable to convert Glu-tRNA(Gln) to Gln-tRNA(Gln) when glutamine was the amide donor.

86 otransferase to convert Glu-tRNA(Gln) to Gln-tRNA(Gln).

87 idotransferase converts Glu-tRNA(Gln) to Gln-tRNA(Gln).

88 e GatDE converts this mischarged tRNA to Gln-tRNA(Gln).

89 Archaea make glutaminyl-tRNA (Gln-tRNA(Gln)) in a two-step process; a non-discriminating g

90 The amide aminoacyl-tRNAs, Gln-tRNA(Gln) and Asn-tRNA(Asn), are formed in many bacteria

91 means for formation of correctly charged Gln-tRNAGln through the transamidation of misacylated Glu-tR

92 ck that matches translational demand for Gln-tRNAGln to aaRS recharging capacity.

93 yme is essential for the biosynthesis of Gln-tRNAGln, an obligate intermediate in translation.

94 at transamidation is the only pathway to Gln-tRNAGln in B. subtilis and that glutamyl-tRNAGln amidotr

95 the RNA component of the contemporary GlnRS-tRNA(Gln) complex in mediating amino acid specificity.

96 structures of unliganded GlnRS and the GlnRS-tRNA(Gln) complex reveal that the Glu34 and Glu73 side c

97 he intermediate indicates that GatE is a Glu-tRNA(Gln) kinase.

98 In the absence of the amido acceptor, Glu-tRNA(Gln), the enzyme has basal glutaminase activity tha

99 otransferase converted Asp-tRNA(Asn) and Glu-tRNA(Gln) into Asn-tRNA and Gln-tRNA, respectively.

100 ify Asp-tRNA(Asn) into Asn-tRNA(Asn) and Glu-tRNA(Gln) into Gln-tRNA(Gln); (iv) the TonB receptors an

101 e enzyme transamidates Asp-tRNA(Asn) and Glu-tRNA(Gln) with similar efficiency (k(cat)/K(m) of 1368.4

102 of ATP hydrolysis requires both Gln and Glu-tRNA(Gln).

103 apable of forming both Glu-tRNA(Glu) and Glu-tRNA(Gln).

104 by the glutamine-dependent Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase (Asp/Glu-Adt).

105 aminase but only in the presence of both Glu-tRNA(Gln) and the other subunit, GatE.

106 In contrast, Glu-tRNA(Gln) stimulates basal ATP hydrolysis slightly, but

107 specialized amidotransferase to convert Glu-tRNA(Gln) to Gln-tRNA(Gln) needed for protein synthesis.

108 yze glutamine and were unable to convert Glu-tRNA(Gln) to Gln-tRNA(Gln) when glutamine was the amide

109 taminyl-tRNA amidotransferase to convert Glu-tRNA(Gln) to Gln-tRNA(Gln).

110 nstrated that recombinant GatAB converts Glu-tRNA(Gln) to Gln-tRNA(Gln) in vitro.

111 A(Gln), and an amidotransferase converts Glu-tRNA(Gln) to Gln-tRNA(Gln).

112 luRS2 only misacylates tRNA(Gln) to form Glu-tRNA(Gln).

113 s primarily synthesized by first forming Glu-tRNA(Gln), followed by conversion to Gln-tRNA(Gln) by a

114 lutamyl-tRNA synthetase (ND-GluRS) forms Glu-tRNA(Gln), while the heterodimeric amidotransferase GatD

115 Gln-tRNA(Gln) is synthesized from Glu-tRNA(Gln) in most microorganisms by a tRNA-dependent ami

116 However, Glu-tRNA(Gln) activates the glutaminase activity of the enzy

117 the proposed high energy intermediate in Glu-tRNA(Gln) transamidation.

118 g the misaminoacylated tRNA intermediate Glu-tRNA(Gln).

119 ynthesizes Glu-tRNA(Glu) and cannot make Glu-tRNA(Gln).

120 NA(Asn) by conversion of the misacylated Glu-tRNA(Gln) and Asp-tRNA(Asn) species catalyzed by the Gat

121 hia coli GlnRS to synthesize misacylated Glu-tRNA(Gln) by 16,000-fold.

122 production of Gln-tRNA(Gln): misacylated Glu-tRNA(Gln) is transamidated by a Gln-dependent amidotrans

123 e produce Gln-tRNA(Gln) from misacylated Glu-tRNA(Gln) through the transamidation activity of Glu-tRN

124 ential for the production of misacylated Glu-tRNA(Gln).

125 autotrophicus that allows the mischarged Glu-tRNA(Gln) made by the tRNA synthetase to be channeled to

126 ) through the transamidation activity of Glu-tRNA(Gln) amidotransferase (Glu-AdT).

127 s very low in the absence or presence of Glu-tRNA(Gln) and Gln.

128 icus GluRS(ND), which is also capable of Glu-tRNA(Gln) synthesis, now shows that both k(cat) and K(m)

129 The activation of Glu-tRNA(Gln) via gamma-phosphorylation bears a similarity t

130 ydrolysis, ATP hydrolysis, activation of Glu-tRNA(Gln), and aminolysis of activated tRNA by Gln-deriv

131 The fact that only Glu-tRNA(Gln) but not tRNA(Gln) could activate the glutamina

132 amidation of mischarged Asp-tRNA(Asn) or Glu-tRNA(Gln) catalyzed by a heterotrimeric amidotransferase

133 rmation of mis-acylated Asp-tRNA(Asn) or Glu-tRNA(Gln), and the subsequent amidation of these amino a

134 by-product derived from gamma-phosphoryl-Glu-tRNA(Gln), the proposed high energy intermediate in Glu-

135 midation of the mischarged tRNA species, Glu-tRNA(Gln) or Asp-tRNA(Asn).

136 g glutamyl-tRNA synthetase to synthesize Glu-tRNA(Gln) and a glutaminyl-tRNA amidotransferase to conv

137 ered hybrid (GlnRS S1/L1/L2) synthesizes Glu-tRNA(Gln) more than 10(4)-fold more efficiently than Gln

138 NA(Gln) 10(7)-fold more efficiently than Glu-tRNA(Gln) and requires tRNA to synthesize the activated

139 tion), Gln-competitive inhibition of the Glu-tRNA(Gln)/ATP-independent glutaminase activity of Glu-Ad

140 le to transamidate M. thermautotrophicus Glu-tRNA(Gln).

141 ression by Cys-tRNA(Pro), Ser-tRNA(Thr), Glu-tRNA(Gln), and Asp-tRNA(Asn).

142 using the latter product to transamidate Glu-tRNA(Gln) in concert with ATP hydrolysis.

143 icus GatCAB is capable of transamidating Glu-tRNA(Gln) from H. pylori or B. subtilis, and M. thermaut

144 three activities waning in concert when Glu-tRNA(Gln) levels become exhausted.

145 vity by ATP or ATP-gammaS, together with Glu-tRNA(Gln), results either from an allosteric effect due

146 ted evolution of Gln-tRNA synthetase and Glu-tRNAGln amidotransferase, and a novel, class I Lys-tRNA

147 hat GluRS2's primary role is to generate Glu-tRNAGln, not Glu-tRNAGlu.

148 hrough the transamidation of misacylated Glu-tRNAGln, functionally replacing the lack of glutaminyl-t

149 GluRS to produce the required mischarged Glu-tRNAGln substrate.

150 AGln but by a specific transamidation of Glu-tRNAGln.

151 Glutaminyl-tRNA(Gln) and asparaginyl-tRNA(Asn) were likely formed i

152 midation of the mischarged species, glutamyl-tRNA(Gln) and aspartyl-tRNA(Asn), by tRNA-dependent amid

153 ion of the mischarged tRNA species, glutamyl-tRNA(Gln) or aspartyl-tRNA(Asn).

154 ional unit of the Bacillus subtilis glutamyl-tRNAGln amidotransferase have been cloned.

155 Gln-tRNAGln in B. subtilis and that glutamyl-tRNAGln amidotransferase is a novel and essential compon

156 NPH-I, the viral core protein E11, and host tRNA(Gln).

157 st adopt the hairpinned conformation seen in tRNA(Gln) complexed with its synthetase.

158 ing 9, 21, and 59 in tRNA(Cys) with those in tRNA(Gln) did not construct a functional core that conta

159 mJ target for Am in tRNA(Pro(GGG)) and Um in tRNA(Gln(UUG)) by mass spectrometric analysis.

160 ously been determined for the nucleotides in tRNAGln.

161 tion of the weak first (U1-A72) base pair in tRNAGln by stacking between A72 and G2.

162 Instead, tRNA(Gln) is initially acylated with glutamate by glutam

163 In contrast, GluRS2 only misacylates tRNA(Gln) to form Glu-tRNA(Gln).

164 ylation of mt-tRNA(Glu), mt-tRNA(Lys) and mt-tRNA(Gln).

165 duced by two to tenfold compared with native tRNA(Gln), consistent with previous findings that the te

166 Neither tRNA(Glu) nor tRNA(Gln) were substrates.

167 The fact that only Glu-tRNA(Gln) but not tRNA(Gln) could activate the glutaminase activity of Gat

168 their specific interactions with both A76 of tRNA(Gln)++ and glutamine.

169 n via the invariant 3'-terminal adenosine of tRNA(Gln).

170 d total translation, the reduced charging of tRNA(Gln) in amino-acid-deprived cells also leads to spe

171 imately 10-20% of the cellular complement of tRNA(Gln)(UUG) is present in mitochondrial RNA fractions

172 otides at the acceptor and anticodon ends of tRNA(Gln) produced a tRNA substrate which was efficientl

173 robustly aminoacylates tRNA(Glu1) instead of tRNA(Gln).

174 , and has no major effect on the kinetics of tRNA(Gln) glutamylation nor transamidation.

175 ndence of a G15-G48 Levitt pair, a number of tRNA(Gln) species containing G15-G48 were constructed an

176 how GluRS2 achieves specific recognition of tRNA(Gln) while rejecting the two H. pylori tRNA(Glu) is

177 Reexamination of the identity set of tRNA(Gln) in the light of these results indicates that i

178 Finally, when marked versions of tRNA(Gln)(UUG) and tRNA(Gln)(UUA) flanked by identical s

179 c hydrogen bond with U35 in the anticodon of tRNAGln, is involved in initial RNA recognition and is a

180 located in the anticodon and acceptor end of tRNAGln described previously.

181 A is not formed by direct glutaminylation of tRNAGln but by a specific transamidation of Glu-tRNAGln.

182 gers a GCN4 response, despite maintenance of tRNAGln charging levels, revealing that normally, the aa

183 The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which natural

184 uncoupling of the first (1.72) base pair of tRNAGln, and tRNAMet was proposed by others to have a si

185 cts with nucleotides in the acceptor stem of tRNAGln, and at R260 in the enzyme's active site were fo

186 H. pylori contains one tRNAGlu and one tRNAGln species, whereas A. ferrooxidans possesses two o

187 uclear and mitochondrial genetic codes, only tRNA(Gln)(UUG) has the capacity to function in mitochond

188 GluRS2 specifically aminoacylating Glu onto tRNA(Gln).

189 R's cis cleavage of precursor tRNA(Gln) (pre-tRNA(Gln)), which lacks certain consensus structures/seq

190 ibit higher fidelity of 5'-maturation of pre-tRNA(Gln) and some of its mutant derivatives.

191 The Km of the T. thermophila enzyme for pre-tRNAGln is 1.6 x 10(-7)M, which is comparable to the val

192 ions containing [4-thio]-uridine-labeled pre-tRNAGln.

193 onents formed cross-link products to the pre-tRNAGln probe.

194 s jannaschii RPR's cis cleavage of precursor tRNA(Gln) (pre-tRNA(Gln)), which lacks certain consensus

195 In addition, pyroglutamyl-tRNA(Gln) accumulated during the reaction catalyzed by t

196 glutamine or glutaminase inhibitors restores tRNA(Gln) charging and the levels of polyglutamine-conta

197 r transient kinetics experiments showed that tRNA(Gln) binds to GlnRS approximately 60-fold weaker wh

198 ary core region of this species contains the tRNA(Gln) G15-C48 pair.

199 zes major identity elements clustered in the tRNA(Gln) acceptor stem.

200 s were identified, defining positions in the tRNA(Gln)(CUG) anticodon stem that restrict first base w

201 mpromised expression of CAG-rich ORFs in the tRNA(Gln)(CUG)-depleted sup70-65 mutant.

202 result different from that reported for the tRNAGln-glutaminyl-tRNA synthetase complex.

203 zing amino acid activation suggests that the tRNAGln-GlnRS complex may be partly analogous to ribonuc

204 se (GluRS) first attaches glutamate (Glu) to tRNA(Gln), and an amidotransferase converts Glu-tRNA(Gln

205 approximately one order of magnitude towards tRNA(Gln).

206 thermophila has three major glutamine tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA).

207 l interdependence, crystal structures of two tRNA(Gln) mutants containing G15-G48 were determined bou

208 GluRS is active toward tRNA(Glu) and the two tRNA(Gln) isoacceptors the organism encodes, but with a

209 glutamine were cocrystallized with wild-type tRNAGln and their structures determined.

210 aturation of tRNA(Trp)(CCA), tRNA(Ile)(UAU), tRNA(Gln)(CUG), tRNA(Lys)(UUU), and tRNA(Val)(CAC).

211 s sequestration capacity, allowing uncharged tRNAGln to interact with Gcn2 kinase.

212 three major glutamine tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA).

213 tRNA synthetase in a quaternary complex with tRNA(Gln), an ATP analog and glutamate reveals that the

214 e (GlnRS) enzyme, which pairs glutamine with tRNA(Gln) for protein synthesis, evolved by gene duplica

215 ficity despite making no hydrogen bonds with tRNAGln.

216 inyl-tRNA synthetase (GlnRS) in complex with tRNAGln and ATP has identified a number a sequence-speci

217 inyl-tRNA synthetase (GlnRS) in complex with tRNAGln, leucine 136 (Leu136) stabilizes the disruption

218 the class I glutaminyl-tRNA synthetase with tRNAGln revealed an uncoupling of the first (1.72) base

219 mitochondrial import; thus sequences within tRNA(Gln)(UUG) direct import.