The 2.2 Å structure of the rRNA methyltransferase ErmC′ and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism

Schluckebier, G.; Zhong, Ping; Stewart, Kent D.; Kavanaugh, Tom J; Abad‐Zapatero, Celerino

doi:10.1006/jmbi.1999.2788

Cited by 98 publications

(140 citation statements)

References 36 publications

Supporting

Mentioning

134

Contrasting

Unclassified

Order By: Relevance

“…The second mutation changes the asparagine residue at position 141 to alanine (N141A) in motif IV, which is highly conserved in rRNA methyltransferases. In the crystal structure of the ErmCЈ rRNA methyltransferase, this amino acid appears to form hydrogen bonds with SAM (3,22). It is also conserved in all of the mtTFB homologs identified thus far (9,19,23).…”

Section: Resultsmentioning

confidence: 97%

“…Our previous analysis of h-mtTFB1 revealed that it is homologous to a family of rRNA methyltransferases, binds the requisite SAM cofactor used by this class of enzymes (19), and methylates a conserved rRNA stem-loop substrate in vivo (24). Sequence comparisons and structural analysis of methyltransferases in general have revealed the presence of eight conserved motifs, designated I to VIII (17,22) (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Human Mitochondrial Transcription Factor B1 Interacts with the C-Terminal Activation Region of h-mtTFA and Stimulates Transcription Independently of Its RNA Methyltransferase Activity

McCulloch

Shadel

2003

Molecular and Cellular Biology

128

View full text Add to dashboard Cite

A significant advancement in understanding mitochondrial gene expression is the recent identification of two new human mitochondrial transcription factors, h-mtTFB1 and h-mtTFB2. Both proteins stimulate transcription in collaboration with the high-mobility group box transcription factor, h-mtTFA, and are homologous to rRNA methyltransferases. In fact, the dual-function nature of h-mtTFB1 was recently demonstrated by its ability to methylate a conserved rRNA substrate. Here, we demonstrate that h-mtTFB1 binds h-mtTFA both in HeLa cell mitochondrial extracts and in direct-binding assays via an interaction that requires the C-terminal tail of h-mtTFA, a region necessary for transcriptional activation. In addition, point mutations in conserved methyltransferase motifs of h-mtTFB1 revealed that it stimulates transcription in vitro independently of S-adenosylmethionine binding and rRNA methyltransferase activity. Furthermore, one mutation (G65A) eliminated the ability of h-mtTFB1 to bind DNA yet did not affect transcriptional activation. These results, coupled with the observation that h-mtTFB1 and human mitochondrial RNA (h-mtRNA) polymerase can also be coimmunoprecipitated, lead us to propose a model in which h-mtTFA demarcates mitochondrial promoter locations and where h-mtTFB proteins bridge an interaction between the C-terminal tail of h-mtTFA and mtRNA polymerase to facilitate specific initiation of transcription. Altogether, these data provide important new insight into the mechanism of transcription initiation in human mitochondria and indicate that the dual functions of h-mtTFB1 can be separated

show abstract

Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Human Mitochondrial Transcription Factor B1 Interacts with the C-Terminal Activation Region of h-mtTFA and Stimulates Transcription Independently of Its RNA Methyltransferase Activity

McCulloch

Shadel

2003

Molecular and Cellular Biology

128

View full text Add to dashboard Cite

show abstract

“…The DNA adenine-N6 methyltransferase M.RsrI has also been cocrystallized with AdoMet and sinefungin; here, too, there were significant differences in the conformations and contacts of the methionine and ornithine components of AdoMet and sinefungin, although the adenine bases of the ligands occupied identical positions (39). The crystal structures of the AdoMet and sinefungin complexes of the rRNA adenine-N6 methyltransferase ErmCЈ highlighted small differences in the conformation of the sinefungin ornithine versus the AdoMet methionine, but significant differences in the protein contacts to the two ligands, including several novel hydrogen bonds to the sinefungin N⑀ atom (40).…”

Section: Structure Of Ecm1 In Complex Withmentioning

confidence: 97%

Mutational Analysis of Encephalitozoon cuniculi mRNA Cap (Guanine-N7) Methyltransferase, Structure of the Enzyme Bound to Sinefungin, and Evidence That Cap Methyltransferase Is the Target of Sinefungin's Antifungal Activity

Zheng¹,

Hausmann²,

Liu

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Cap (guanine-N7) methylation is an essential step in eukaryal mRNA synthesis and a potential target for antiviral, antifungal, and antiprotozoal drug discovery. Previous mutational and structural analyses of Encephalitozoon cuniculi Ecm1, a prototypal cellular cap methyltransferase, identified amino acids required for cap methylation in vivo, but also underscored the nonessentiality of many side chains that contact the cap and AdoMet substrates. Here we tested new mutations in residues that comprise the guaninebinding pocket, alone and in combination. The outcomes indicate that the shape of the guanine binding pocket is more crucial than particular base edge interactions, and they highlight the contributions of the aliphatic carbons of Phe-141 and Tyr-145 that engage in multiple van der Waals contacts with guanosine and S-adenosylmethionine (AdoMet), respectively. We purified 45 Ecm1 mutant proteins and assayed them for methylation of GpppA in vitro. Of the 21 mutations that resulted in unconditional lethality in vivo, 14 reduced activity in vitro to <2% of the wild-type level and 5 reduced methyltransferase activity to between 4 and 9% of wild-type Ecm1. The natural product antibiotic sinefungin is an AdoMet analog that inhibits Ecm1 with modest potency. The crystal structure of an Ecm1-sinefungin binary complex reveals sinefungin-specific polar contacts with main-chain and side-chain atoms that can explain the 3-fold higher affinity of Ecm1 for sinefungin versus AdoMet or S-adenosylhomocysteine (AdoHcy). In contrast, sinefungin is an extremely potent inhibitor of the yeast cap methyltransferase Abd1, to which sinefungin binds 900-fold more avidly than AdoHcy or AdoMet. We find that the sensitivity of Saccharomyces cerevisiae to growth inhibition by sinefungin is diminished when Abd1 is overexpressed. These results highlight cap methylation as a principal target of the antifungal activity of sinefungin.The m 7 GpppN cap structure of eukaryotic messenger RNA is formed by three enzymes. RNA triphosphatase hydrolyzes the 5Ј-triphosphate end of the pre-mRNA to a diphosphate, which is capped with GMP by RNA guanylyltransferase. RNA (guanine-N7) methyltransferase then adds a methyl group from AdoMet 2 to GpppRNA to form m 7 GpppRNA and AdoHcy. This pathway is conserved in all eukaryotic organisms and many eukaryotic viruses (1). The capping enzymes are considered attractive targets for antiviral, antifungal, and antiprotozoal drug discovery (2). Inhibition of cap methylation, in particular, has been touted as an anti-infective strategy based on two lines of evidence: (i) raising the cellular levels of AdoHcy by genetic or pharmacological inhibition of AdoHcy hydrolase blocks replication of many viruses (3, 4), and (ii) the AdoMet analog sinefungin (an inhibitor of cap methylation in vitro) inhibits the growth of diverse viruses, fungi, and protozoan parasites (5-11). Indeed, it was shown recently that sinefungin displays selectivity in inhibiting yeast cap methyltransferases versus the human enzyme in vivo (12).The S...

show abstract

“…The methylation of the N 6 position of adenine has been investigated in DNA (for structural studies see [42][43][44] ) and more recently in rRNA. 45,46 Interestingly, the main catalytic residue in m 6 which is proposed to stabilize the cationic transition state by cation-π interaction. 49,50 And the second one is the aromatic residue Y/F/W of motif VIII, which stabilizes the flipped base outside the DNA helix.…”

Section: Superimposition Of T Thermophilus and M Tuberculosismentioning

confidence: 99%

Crystal Structure of Thermus thermophilus tRNA m1A58 Methyltransferase and Biophysical Characterization of Its Interaction with tRNA

Barraud

Golinelli‐Pimpaneau

Atmanène

et al. 2008

Journal of Molecular Biology

View full text Add to dashboard Cite

The 2.2 Å structure of the rRNA methyltransferase ErmC′ and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism

Cited by 98 publications

References 36 publications

Human Mitochondrial Transcription Factor B1 Interacts with the C-Terminal Activation Region of h-mtTFA and Stimulates Transcription Independently of Its RNA Methyltransferase Activity

Human Mitochondrial Transcription Factor B1 Interacts with the C-Terminal Activation Region of h-mtTFA and Stimulates Transcription Independently of Its RNA Methyltransferase Activity

Mutational Analysis of Encephalitozoon cuniculi mRNA Cap (Guanine-N7) Methyltransferase, Structure of the Enzyme Bound to Sinefungin, and Evidence That Cap Methyltransferase Is the Target of Sinefungin's Antifungal Activity

Crystal Structure of Thermus thermophilus tRNA m1A58 Methyltransferase and Biophysical Characterization of Its Interaction with tRNA

Contact Info

Product

Resources

About