Posttranscriptionally modified nucleosides in transfer RNA: Their locations and frequencies

Grosjean, Henri; Sprinzl, Mathias; Steinberg, Sergey V.

doi:10.1016/0300-9084(96)88117-x

Cited by 133 publications

(111 citation statements)

References 3 publications

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“…To test whether Trm8 and Trm82 proteins are sufficient for m 7 G-methylation of tRNA, we expressed both proteins in E. coli as His6-fusion proteins under control of the P tac promoter+ We find that expression of both proteins in E. coli yields extracts with 100-fold more activity than that in extracts from cells overexpressing either His6-fusion protein alone (data not shown)+ Similarly, copurified His6-Trm8 and His6-Trm82 proteins from cells simultaneously overexpressing both proteins results in preparations with 1,000-fold more activity than that obtained from mock purification or from purification of His6-Trm82 protein alone, and 250-fold more activity than that from purification of His6-Trm8 protein (Fig+ 7)+ We conclude that expression of both Trm8 and Trm82 is sufficient for m 7 G methyltransferase activity, although the 0+4% basal activity produced by Trm8 protein alone suggests that the presence of Trm82 is not an absolute requirement for activity in vitro+ The methyltransferase activity of purified Trm8/Trm82 proteins is the same as that identified as m 7 G by modification of pre-tRNA Phe in crude extracts, as determined by two-dimensional TLC (Fig+ 7C)+ Purified Trm8/Trm82 proteins modify G46 of pre-tRNA Phe to m 7 G Because m 7 G modification of tRNA from different organisms is almost invariably found in the extra loop at G46 (Grosjean et al+, 1995), it seemed highly likely that Trm8/Trm82 proteins were acting at the same site on pre-tRNA Phe + To map the site of m 7 G modification, and to confirm the identity of the modification, we exploited the known sensitivity of m 7 G to borohydride reduction followed by aniline cleavage of the RNA (Wintermeyer & Zachau, 1975)+ After incubation of 39-end-labeled pre-tRNA Phe with Trm8/Trm82 proteins, aniline cleavage yielded a single band (Fig+ 8)+ Comparison of the size of the cleaved product with a guanine cleavage ladder generated from pre-tRNA Phe (Peattie, 1979) demonstrates that cleavage occurs at G46, the same site that is modified to m 7 G in vivo+ Since m 7 G is the only common modified guanine nucleotide that is sensitive to aniline, this experiment also provides further indication that the modification formed by Trm8/Trm82 is m 7 G+…”

Section: Production Of Both Trm8 and Trm82 Proteins Is Sufficient Formentioning

confidence: 83%

Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA

Alexandrov

Martzen

Phizicky

2002

RNA

266

271

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ABSTRACT7-methylguanosine (m 7 G) modification of tRNA occurs widely in eukaryotes and bacteria, is nearly always found at position 46, and is one of the few modifications that confers a positive charge to the base. Screening of a Saccharomyces cerevisiae genomic library of purified GST-ORF fusion proteins reveals two previously uncharacterized proteins that copurify with m 7 G methyltransferase activity on pre-tRNA Phe . ORF YDL201w encodes Trm8, a protein that is highly conserved in prokaryotes and eukaryotes and that contains an S-adenosylmethionine binding domain. ORF YDR165w encodes Trm82, a less highly conserved protein containing putative WD40 repeats, which are often implicated in macromolecular interactions. Neither protein has significant sequence similarity to yeast Abd1, which catalyzes m 7 G modification of the 59 cap of mRNA, other than the methyltransferase motif shared by Trm8 and Abd1. Several lines of evidence indicate that both Trm8 and Trm82 proteins are required for tRNA m 7 G-methyltransferase activity: Extracts derived from strains lacking either gene have undetectable m 7 G methyltransferase activity, RNA from strains lacking either gene have much reduced m 7 G, and coexpression of both proteins is required to overproduce activity. Aniline cleavage mapping shows that Trm8/Trm82 proteins modify pre-tRNA Phe at G46, the site that is modified in vivo. Trm8 and Trm82 proteins form a complex, as affinity purification of Trm8 protein causes copurification of Trm82 protein in approximate equimolar yield. This functional two-protein family appears to be retained in eukaryotes, as expression of both corresponding human proteins, METTL1 and WDR4, is required for m 7 Gmethyltransferase activity.

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Section: Production Of Both Trm8 and Trm82 Proteins Is Sufficient Formentioning

confidence: 83%

Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA

Alexandrov

Martzen

Phizicky

2002

RNA

266

271

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“…This modification is found in all three major phylogenetic domains and is one of the most common RNA modifications, comprising z8% of all tRNA modifications (366/4781) (Grosjean et al 1995;Sprinzl et al 1998), as well as a large fraction of the modifications found in rRNA and snRNA Fournier 2002, 2003). Formation of 29-O-methylated residues is effected by either of two methods.…”

Section: Introductionmentioning

confidence: 99%

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

Wilkinson¹,

Crary²,

Jackman³

et al. 2007

RNA

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The methylation of the ribose 29-OH of RNA occurs widely in nature and in all stable RNAs and occurs at five positions in yeast tRNA. 29-O-methylation of tRNA at position 4 is interesting because it occurs in the acceptor stem (which is normally undermodified), it is the only 29-O-methylation that occurs in the middle of a duplex region in tRNA, the modification is conserved in eukaryotes, and the features of the tRNA necessary for substrate recognition are poorly defined. We show here that Saccharomyces cerevisiae ORF YOL125w (TRM13) is necessary and sufficient for 29-O-methylation at position 4 of yeast tRNA. Biochemical analysis of the S. cerevisiae proteome shows that Trm13 copurifies with 29-O-methylation activity, using tRNA Gly(GCC) as a substrate, and extracts made from a trm13-D strain have undetectable levels of this activity. Trm13 is necessary for activity in vivo because tRNAs isolated from a trm13-D strain lack the corresponding 29-O-methylated residue for each of the three known tRNAs with this modification. Trm13 is sufficient for 29-O-methylation at position 4 in vitro since yeast Trm13 protein purified after expression in Escherichia coli has the same activity as that produced in yeast. Trm13 protein binds substrates tRNA His and tRNA Gly(GCC) with K D values of 85 6 8 and 100 6 14 nM, respectively, and has a K M for tRNA His of 10 nM, but binds nonsubstrate tRNAs very poorly (K D > 1 mM). Trm13 is conserved in eukaryotes, but there is no sequence similarity between Trm13 and other known methyltransferases.

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“…Nucleoside modification is perhaps the most elaborate step in tRNA processing (Bjö rk, 1995). Over 90 modified nucleosides have been characterized (McCloskey & Crain, 1998), many of which are conserved across broad phylogenetic boundaries (Grosjean et al, 1995). Their modifications vary from simple methylation of the base or ribose ring to extensive hypermodification of the canonical bases, resulting in structural changes requiring multiple enzymatic steps.…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary X-ray characterization of the nitrile reductase QueF: a queuosine-biosynthesis enzyme

et al. 2005

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QueF (MW = 19.4 kDa) is a recently characterized nitrile oxidoreductase which catalyzes the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQ 0 ) to 7-aminomethyl-7-deazaguanine, a late step in the biosynthesis of the modified tRNA nucleoside queuosine. Initial crystals of homododecameric Bacillus subtilis QueF diffracted poorly to 8.0 Å . A three-dimensional model based on homology with the tunnel-fold enzyme GTP cyclohydrolase I suggested catalysis at intersubunit interfaces and a potential role for substrate binding in quaternary structure stabilization. Guided by this insight, a second crystal form was grown that was strictly dependent on the presence of preQ 0 . This crystal form diffracted to 2.25 Å resolution.

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Posttranscriptionally modified nucleosides in transfer RNA: Their locations and frequencies

Cited by 133 publications

References 3 publications

Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA

Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

Crystallization and preliminary X-ray characterization of the nitrile reductase QueF: a queuosine-biosynthesis enzyme

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