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S-adenosyl-L-methionine + 5-carboxymethylaminomethyluridine34 in tRNA
S-adenosyl-L-homocysteine + 5-carboxymethylaminomethyl-2'-O-methyluridine34 in tRNA
S-adenosyl-L-methionine + 5-carboxymethylaminomethyluridine34 in tRNALeu
S-adenosyl-L-homocysteine + 5-carboxymethylaminomethyl-2'-O-methyluridine34 in tRNALeu
S-adenosyl-L-methionine + cytidine34 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNA
S-adenosyl-L-methionine + cytidine34 in tRNALeuCAA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuCAA
S-adenosyl-L-methionine + cytidine34 in tRNALeuUAA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuUAA
additional information
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S-adenosyl-L-methionine + 5-carboxymethylaminomethyluridine34 in tRNA
S-adenosyl-L-homocysteine + 5-carboxymethylaminomethyl-2'-O-methyluridine34 in tRNA
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TrmL catalyzes the methyl transfer from SAM to the 2'-OH of the wobble nucleotide in tRNALeu cmnm5UmAA, which decodes codons UUA and UUG in the Phe/Leu mixed box, and tRNALeu CmAA, which reads the UUG codon included in the Leu family box
o.e. tRNALeucmnm5UmAA
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S-adenosyl-L-methionine + 5-carboxymethylaminomethyluridine34 in tRNA
S-adenosyl-L-homocysteine + 5-carboxymethylaminomethyl-2'-O-methyluridine34 in tRNA
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TrmL catalyzes the methyl transfer from SAM to the 2'-OH of the wobble nucleotide in tRNALeu cmnm5UmAA, which decodes codons UUA and UUG in the Phe/Leu mixed box, and tRNALeu CmAA, which reads the UUG codon included in the Leu family box. The wobble nucleoside in tRNALeu cmnm5UmAA and tRNALeuCmAA is not thiolated. Nevertheless methylation of the 2'-hydroxyl group favors the C3'-endo ribose conformation for all nucleosides, although the effect is more marked with pyrimidines
o.e. tRNALeucmnm5UmAA
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S-adenosyl-L-methionine + 5-carboxymethylaminomethyluridine34 in tRNALeu
S-adenosyl-L-homocysteine + 5-carboxymethylaminomethyl-2'-O-methyluridine34 in tRNALeu
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S-adenosyl-L-methionine + 5-carboxymethylaminomethyluridine34 in tRNALeu
S-adenosyl-L-homocysteine + 5-carboxymethylaminomethyl-2'-O-methyluridine34 in tRNALeu
the enzyme methylates the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNALeu(CmAA) and tRNALeu(cmnm5UmAA)
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S-adenosyl-L-methionine + cytidine34 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNA
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S-adenosyl-L-methionine + cytidine34 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNA
the enzyme methylates the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNALeu(CmAA) and tRNALeu(cmnm5UmAA)
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S-adenosyl-L-methionine + cytidine34 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNA
the enzyme methylates the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNALeu(CmAA) and tRNALeu(cmnm5UmAA). No methylation of fully synthetic in vitro transcrips of tRNA(CAA), lacking all natural modifications present in in vivo transcripts. The motifs in tRNALeu required for YibK recognition and catalysis include the N6-(isopentenyl)-2-methylthioadenosine (ms2i6A) at position 37 and a pyridine at position 34. It has a clear preference for adenosine at position 35
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S-adenosyl-L-methionine + cytidine34 in tRNALeuCAA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuCAA
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S-adenosyl-L-methionine + cytidine34 in tRNALeuCAA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuCAA
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S-adenosyl-L-methionine + cytidine34 in tRNALeuCAA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuCAA
diverse mutant variants of tRNALeuCAA, overview
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S-adenosyl-L-methionine + cytidine34 in tRNALeuCAA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuCAA
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S-adenosyl-L-methionine + cytidine34 in tRNALeuUAA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuUAA
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S-adenosyl-L-methionine + cytidine34 in tRNALeuUAA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuUAA
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S-adenosyl-L-methionine + cytidine34 in tRNALeuUAA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuUAA
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additional information
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in addition to a pyrimidine at position 34, the enzyme requires the presence of N6-(isopentenyl)-2-methylthioadenosine (ms2i6A) at position 37 as a positive identity determinant. Modification i6A, produced by MiaA, is enough to promote recognition of the substrate tRNAs by TrmL. Recognition of an amber codon by the supP suppressor, which is a mutant tRNALeu CmAA carrying the A35U change in the anticodon, is impaired if the suppressor lacks the 2'-O-methylation at the wobble position
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additional information
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anticodon stem-loop minihelices with an extension of 2 base pairs are the minimal substrate for EcTrmL methylation. A35 is a key residue for TrmL recognition, while A36-A37-A38 are important either via direct interaction with TrmL or due to the necessity for prior isopentenylation (i6) at A37. In addition, TrmL only methylates pyrimidines but not purine residues at the wobble position, and the 20-O-methylation relies on prior N6-isopentenyladenosine modification at position 37. tRNALeuCAA and tRNALeuUAA isoacceptors are the only two RNA substrates of TrmL. i6A37 is sufficient to restore 20-O-methylation at C34 of EctRNALeuCAA transcripts in vitro. TrmL activity is sensitive to the modified base at position 37, a defect in synthesis of ms2i6A37 at position 37, leads to the loss of 20-O-methylation at C/U 34 at tRNALeuCAA and tRNALeuUAA isoacceptors.16 The isopentenyl modification at position 37 of transcribed tRNALeu is sufficient to recruit TrmL for methylation of nucleotides C34/U34. Although the affinity of i6A-Ts-tRNALeu CAA to EcTrmL is lower than for wild-type EctRNALeuCAA, the i6A modification has a consequent effect on the binding strength of tRNA transcripts for EcTrmL
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additional information
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the enzyme TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to tRNALeuCAA and tRNALeuUAA isoacceptors without the involvement of other tRNA-binding proteins. EcTrmL alone can efficiently methylate both tRNALeuCAA and tRNALeuUAA isoacceptors by using in vivo-purified tRNA substrates with certain modifications, but not unmodified tRNALeu
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additional information
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the C/U34m modification occurs in leucine tRNACmAA and tRNAcmnm5UmAA. Both of these leucine tRNA isoacceptors recognize UUA-Leu and UUG-Leu codons, and also carry the i6A37 tRNA modification that assists in the optimal decoding of the Leu codons during rpoS translation
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additional information
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the C/U34m modification occurs in leucine tRNACmAA and tRNAcmnm5UmAA. Both of these leucine tRNA isoacceptors recognize UUA-Leu and UUG-Leu codons, and also carry the i6A37 tRNA modification that assists in the optimal decoding of the Leu codons during rpoS translation
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additional information
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the enzyme TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to tRNALeuCAA and tRNALeuUAA isoacceptors without the involvement of other tRNA-binding proteins. EcTrmL alone can efficiently methylate both tRNALeuCAA and tRNALeuUAA isoacceptors by using in vivo-purified tRNA substrates with certain modifications, but not unmodified tRNALeu
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S-adenosyl-L-methionine + 5-carboxymethylaminomethyluridine34 in tRNA
S-adenosyl-L-homocysteine + 5-carboxymethylaminomethyl-2'-O-methyluridine34 in tRNA
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TrmL catalyzes the methyl transfer from SAM to the 2'-OH of the wobble nucleotide in tRNALeu cmnm5UmAA, which decodes codons UUA and UUG in the Phe/Leu mixed box, and tRNALeu CmAA, which reads the UUG codon included in the Leu family box
o.e. tRNALeucmnm5UmAA
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S-adenosyl-L-methionine + 5-carboxymethylaminomethyluridine34 in tRNALeu
S-adenosyl-L-homocysteine + 5-carboxymethylaminomethyl-2'-O-methyluridine34 in tRNALeu
S-adenosyl-L-methionine + cytidine34 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNA
S-adenosyl-L-methionine + cytidine34 in tRNALeuCAA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuCAA
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S-adenosyl-L-methionine + cytidine34 in tRNALeuUAA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNALeuUAA
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S-adenosyl-L-methionine + 5-carboxymethylaminomethyluridine34 in tRNALeu
S-adenosyl-L-homocysteine + 5-carboxymethylaminomethyl-2'-O-methyluridine34 in tRNALeu
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S-adenosyl-L-methionine + 5-carboxymethylaminomethyluridine34 in tRNALeu
S-adenosyl-L-homocysteine + 5-carboxymethylaminomethyl-2'-O-methyluridine34 in tRNALeu
the enzyme methylates the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNALeu(CmAA) and tRNALeu(cmnm5UmAA)
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S-adenosyl-L-methionine + cytidine34 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNA
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S-adenosyl-L-methionine + cytidine34 in tRNA
S-adenosyl-L-homocysteine + 2'-O-methylcytidine34 in tRNA
the enzyme methylates the ribose at the nucleotide 34 wobble position in the two leucyl isoacceptors tRNALeu(CmAA) and tRNALeu(cmnm5UmAA)
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evolution
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TrmL is a representative protein of SPOUTenzymes, a class of S-adenosyl-L-methionine-dependent methyltransferases that exhibit an unusual fold with a very deep topological knot. TrmL is one of the smallest alpha/beta-knot proteins
evolution
the enzyme belongs to the SPOUT tRNA MTase superfamily
evolution
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TrmL is a member of the SPOUT superfamily. TrmH, TrmJ and TrmL belong to the SpoU family, and TrmD belongs to the TrmD family. Unlike other transfer RNAs (tRNA)-modifying enzymes from the SPOUT methyltransferase superfamily, the tRNA (Um34/Cm34) methyltransferase TrmL lacks the usual extension domain for tRNA binding and consists only of a SPOUT domain. Domain architectures of SPOUT tRNA MTases and the sequence alignment of TrmLs, overview. TrmL enzymes are widely distributed throughout the bacterial kingdom
evolution
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TrmL is a member of the SPOUT superfamily. TrmH, TrmJ and TrmL belong to the SpoU family, and TrmD belongs to the TrmD family. Unlike other transfer RNAs (tRNA)-modifying enzymes from the SPOUT methyltransferase superfamily, the tRNA (Um34/Cm34) methyltransferase TrmL lacks the usual extension domain for tRNA binding and consists only of a SPOUT domain. Domain architectures of SPOUT tRNA MTases and the sequence alignment of TrmLs, overview. TrmL enzymes are widely distributed throughout the bacterial kingdom
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malfunction
inactivation of yibK leads to loss of 2'-O-methylation at position 34 in both tRNALeu(CmAA) and tRNALeu(cmnm5UmAA). Loss of YibK methylation reduces the efficiency of codonwobble base interaction. Inactivation of yibK has no detectable effect on steady-state growth rate, although a distinct disadvantage is noted in multiple-round, mixed-population growth experiments, suggesting that the ability to recover from the stationary phase is impaired. Methylation is restored in vivo by complementing with a recombinant copy of yibK
malfunction
the defect in RpoS translation in the absence of i6A37 prenyl transferase (MiaA) is due to the inability to add the C/U34m modification to UUX-Leu tRNAs
metabolism
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modifications at the wobble uridine, U34, of tRNAs reading two degenerate codons ending in purine are complex and result from the activity of two multi-enzyme pathways, the IscSeMnmA and MnmEG pathways, which independently work on positions 2 and 5 of the U34 pyrimidine ring, respectively, and from a third single-step pathway, controlled by TrmL, i.e. YibK, that modifies the 2'-hydroxyl group of the ribose. TrmL occurs as a late step in the maturation of the tRNALeu isoacceptors
metabolism
role of several tRNA modifications on rpoS or hfq expression: 2'-O-methylation of cytidine or uridine, at the wobble position (C/Um), 2-thiouridine at the wobble position (s2U), as well as isopentenyl adenosine 37(i6A37), overview
physiological function
TrmL is the prokaryotic methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the wobble base of tRNALeuCAA and tRNALeuUAA isoacceptors. This Cm34/Um34 modification affects codon-anticodon interactions and is essential for translational fidelity
physiological function
the addition of the 2'-O-methylcytidine/uridine 34 (C/U34m) tRNA modification by tRNA (cytidine/uridine-2'O)-ribose methyltransferase L (TrmL) in Escherichia coli requires the presence of the N6-isopentenyl adenosine 37 (i6A37). The C/U34m and s2U34 tRNA modifications are necessary for full proper rpoS translation. Gene trmL is necessary for leucine decoding during RpoS expression. The mechanism of action of the i6A37 tRNA modification on RpoS expression is, at least in part, promotion of efficient UUX-leucine decoding. Possibly the TrmL-catalyzed C/U34m tRNA modification is dispensable for hfq translation, while the MiaA catalyzed i6A37 is necessary for efficient hfq translation
additional information
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the enzyme TrmL functions as a homodimer by using the conserved C-terminal half of the SPOUT domain for catalysis, whereas residues from the less-conserved N-terminal half of the other subunit participate in tRNA recognition. Analysis of the structure of the active site. EcTrmL dimer formation is essential for tRNA recognition. The residue Y142 is critical for maintaining the dimeric form of EcTrmL, which is consistent with its central position at the interface
additional information
TrmL-catalyzed 2'-O-methylation requires its homodimerization. TrmL exhibits a fine-tuned tRNA substrate recognition mechanism. The variable arm of tRNA is not a recognition element for EcTrmL. The anticodon stem of tRNA does not contain recognition elements for EcTrmL
additional information
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the enzyme TrmL functions as a homodimer by using the conserved C-terminal half of the SPOUT domain for catalysis, whereas residues from the less-conserved N-terminal half of the other subunit participate in tRNA recognition. Analysis of the structure of the active site. EcTrmL dimer formation is essential for tRNA recognition. The residue Y142 is critical for maintaining the dimeric form of EcTrmL, which is consistent with its central position at the interface
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K42E
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site-directed mutagenesis, inactive mutant
K81E
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
R104E
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
R129E
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site-directed mutagenesis, inactive mutant
R20E
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site-directed mutagenesis, inactive mutant
R28E
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site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme
R43E
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site-directed mutagenesis, inactive mutant
R45E
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site-directed mutagenesis, inactive mutant
R46E
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site-directed mutagenesis, inactive mutant
R59E
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site-directed mutagenesis, inactive mutant
R64E
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
R74E
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Y142A
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site-directed mutagenesis, the mutant is a monomer in contrast to the wild-type enzyme. Mutant Y142A fails to form a complex with tRNA
K42E
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site-directed mutagenesis, inactive mutant
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K81E
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
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R46E
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site-directed mutagenesis, inactive mutant
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R59E
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site-directed mutagenesis, inactive mutant
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additional information
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construction of gene trmL deletion mutants
additional information
generation of trmL+ and trmL- PBAD-rpoS990-lacZ translational fusions (in rssB- backgrounds) cells, executed for PBAD-rpoS990-lacZ translational fusions with rpoS UUA-Leu CUX-Leu mutations (leu*1)-strains KMT36002 and KMT36010, for rpoS UUG-Leu to CUX-leu mutation (leu*2)-strains KMT37002 and KMT37010, and for rpoS with both UUA-Leu to CUX-Leu and UUG-Leu to CUX-Leu mutations (leu*3)-strains KMT33001 and KMT33013, overview. Comparison of expression of PBAD-rpoS990-lacZ translational fusions carrying wild-type UUX leucine codons with derivatives in which UUX codons are changed to CUX codons, in the presence and absence of TrmL or TusA. The absence of these proteins, and therefore presumably the modifications they catalyze, both abolish PBAD-rpoS990-lacZ translation activity. UUX-Leu to CUX-Leu codon mutations in rpoS suppress the trmL requirement for PBAD-rpoS990-lacZ expression
additional information
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generation of trmL+ and trmL- PBAD-rpoS990-lacZ translational fusions (in rssB- backgrounds) cells, executed for PBAD-rpoS990-lacZ translational fusions with rpoS UUA-Leu CUX-Leu mutations (leu*1)-strains KMT36002 and KMT36010, for rpoS UUG-Leu to CUX-leu mutation (leu*2)-strains KMT37002 and KMT37010, and for rpoS with both UUA-Leu to CUX-Leu and UUG-Leu to CUX-Leu mutations (leu*3)-strains KMT33001 and KMT33013, overview. Comparison of expression of PBAD-rpoS990-lacZ translational fusions carrying wild-type UUX leucine codons with derivatives in which UUX codons are changed to CUX codons, in the presence and absence of TrmL or TusA. The absence of these proteins, and therefore presumably the modifications they catalyze, both abolish PBAD-rpoS990-lacZ translation activity. UUX-Leu to CUX-Leu codon mutations in rpoS suppress the trmL requirement for PBAD-rpoS990-lacZ expression
additional information
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construction of gene trmL deletion mutants
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Benitez-Paez, A.; Villarroya, M.; Douthwaite, S.; Gabaldon, T.; Armengod, M.E.
YibK is the 2'-O-methyltransferase TrmL that modifies the wobble nucleotide in Escherichia coli tRNA(Leu) isoacceptors
RNA
16
2131-2143
2010
Escherichia coli (P0AGJ7)
brenda
Armengod, M.E.; Moukadiri, I.; Prado, S.; Ruiz-Partida, R.; Benitez-Paez, A.; Villarroya, M.; Lomas, R.; Garzon, M.J.; Martinez-Zamora, A.; Meseguer, S.; Navarro-Gonzalez, C.
Enzymology of tRNA modification in the bacterial MnmEG pathway
Biochimie
94
1510-1520
2012
Escherichia coli
brenda
Liu, R.J.; Zhou, M.; Fang, Z.P.; Wang, M.; Zhou, X.L.; Wang, E.D.
The tRNA recognition mechanism of the minimalist SPOUT methyltransferase, TrmL
Nucleic Acids Res.
41
7828-7842
2013
Escherichia coli, Escherichia coli MT102
brenda
Zhou, M.; Long, T.; Fang, Z.P.; Zhou, X.L.; Liu, R.J.; Wang, E.D.
Identification of determinants for tRNA substrate recognition by Escherichia coli C/U34 2-O-methyltransferase
RNA Biol.
12
900-911
2015
Escherichia coli (P0AGJ7)
brenda
Aubee, J.I.; Olu, M.; Thompson, K.M.
TrmL and TusA are necessary for rpoS and MiaA is required for hfq expression in Escherichia coli
Biomolecules
7
39
2017
Escherichia coli (P0AGJ7), Escherichia coli
brenda