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 s and use either tRNAGlu or both tRNAGlu and tRNAGln.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

45 ognate tRNAs but to a decreased affinity for tRNAGln.

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

47 AGlu, whereas GluRS2 was specific solely for tRNAGln.

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

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

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

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

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

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

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

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

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

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

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

59 2 nM) sequesters the tRNA synthetase for Gln-tRNA(Gln) formation, with GatDE reducing the affinity of

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

155 ously been determined for the nucleotides in tRNAGln.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

181 GluRS2 specifically aminoacylating Glu onto tRNA(Gln).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

206 ficity despite making no hydrogen bonds with tRNAGln.

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

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

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

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