A Unique RNA Fold in the RumA-RNA-Cofactor Ternary Complex Contributes to Substrate Selectivity and Enzymatic Function

Lee, Tom T.; Agarwalla, Sanjay; Stroud, Rhonda M.

doi:10.1016/j.cell.2004.12.037

Cited by 100 publications

(159 citation statements)

References 47 publications

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“…The strategy of using an RNA base rather than an amino acid to stabilize the RNA core after base flipping is identical to the strategy used in the RumA-AdoHcy-RNA ternary complex (8). Further, the nonsequential stack (53-58-57-56-55) is surprisingly similar to the stack formed by the RumA substrate bases 1938-1942-1941-1940 (Fig.…”

Section: Trma and Ruma Have The Same Folds But Only The Catalytic Corementioning

confidence: 68%

“…tRNA U54 methylation by TrmA requires refolding of the T loop into a conformation that features a nonsequential collinear stack of bases, similar to that formed by the RNA bound to the active site of the m 5 U MTase RumA (8). In both enzymes, refolding of a target-containing loop segment into this base-stacked conformation displays a U-U-C sequence (where the first U is the target) to the active-site cavity.…”

Section: Resultsmentioning

confidence: 99%

“…In many AdoMet-dependent MTases, the adenine ring of the cofactor interdigitates between two aromatic side chains. In RumA an RNA base from the substrate flips into the AdoMet site and substitutes for one of these residues, thus forming part of the AdoMet binding site (8). Tyr-218 in TrmA could pack against one side of the adenine ring of the bound cofactor.…”

Section: Resultsmentioning

confidence: 99%

“…Van der Waals, aromatic stacking, and hydrogenbond interactions stabilize the target base, U54, in a productive orientation. All of these interactions are conserved in RumA (8).…”

Section: Contacts With the Consensus Fold Are Highly Conserved Betweementioning

confidence: 99%

“…The guanidinium group of Arg-302 (Arg-366/RumA) forms hydrogen bonds to O2Ј and O3Ј of U54. The U54 base is hydrogen-bonded to the side chains of three conserved residues with likely roles in catalysis: Gln-190 (Gln-265/ RumA), Asp-299 (Asp-363/RumA), and Gln-358 (Glu-358 in wildtype TrmA, Glu-424/RumA) (8,16).…”

Section: Contacts With the Consensus Fold Are Highly Conserved Betweementioning

confidence: 99%

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Structure of a TrmA–RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m ⁵ U methyltransferases

Alian¹,

Lee²,

Griner³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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100

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TrmA catalyzes S-adenosylmethionine (AdoMet)-dependent methylation of U54 in most tRNAs. We solved the structure of the Escherichia coli 5-methyluridine (m 5 U) 54 tRNA methyltransferase (MTase) TrmA in a covalent complex with a 19-nt T arm analog to 2.4-Å resolution. Mutation of the TrmA catalytic base Glu-358 to Gln arrested catalysis and allowed isolation of the covalent TrmA-RNA complex for crystallization. The protein-RNA interface includes 6 nt of the T loop and two proximal base pairs of the stem. U54 is flipped out of the loop into the active site. A58 occupies the space of the everted U54 and is part of a collinear base stack G53-A58 -G57-C56 -U55. The RNA fold is different from T loop conformations in unbound tRNA or T arm analogs, but nearly identical to the fold of the RNA loop bound at the active site of the m 5 U MTase RumA. In both enzymes, this consensus fold presents the target U and the following two bases to a conserved binding groove on the protein. Outside of this fold, the RumA and TrmA substrates have completely different structures and protein interfaces. Loop residues other than the target U54 make more than half of their hydrogen bonds to the protein via sugar-phosphate moieties, accounting, in part, for the broad consensus sequence for TrmA substrates.RNA modification ͉ substrate specificity ͉ tRNA ͉ x-ray crystallography P osttranscriptional modification of noncoding RNA occurs in all kingdoms of life. These modifications can alter the physical and/or chemical properties of the nucleotides. Several such modifications are clustered at functionally important sites on tRNA and rRNA and have been shown to contribute to the fidelity and efficiency of mRNA translation (1-3).S-Adenosylmethionine (AdoMet)-dependent methylation of uridine to 5-methyluridine (m 5 U) occurs at two sites in the Escherichia coli ribosome and at a single site in E. coli tRNAs. A different enzyme is responsible for modification in each of these three environments. TrmA (formerly known as RUMT) modifies U54 in the T arms of most tRNAs (4). RumA and RumB modify U1939 (5) and U747 (6), respectively, of 23S RNA. TrmA, RumA, and RumB are two-or three-domain proteins that have little overall sequence homology, but contain six conserved sequence motifs that are indicative of a class I AdoMet-dependent MTase fold (7). The chemical mechanism of m 5 U54 methylation involves Michael addition of a catalytic cysteine to C6 of the uridine (Fig. 1A) (4).The first reported crystal structure of an m 5 U MTase-RNA complex was that of a ternary complex formed with RumA, AdoMet, and a 37-nt fragment of 23S RNA, (bases 1932-1968) (8).The C5-hydrogen of the target, U1939, in this fragment had been substituted with fluorine, which stalled the methylation reaction at the proton abstraction step, forming a stable covalent complex (presumed to be an analog to intermediate 3 in Fig. 1 A). Based on the structure, the invariant Glu-424 was proposed to be the general base in the reaction, and this hypothesis was confirmed by mutagenesis (8). T...

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Section: Trma and Ruma Have The Same Folds But Only The Catalytic Corementioning

confidence: 68%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…Van der Waals, aromatic stacking, and hydrogenbond interactions stabilize the target base, U54, in a productive orientation. All of these interactions are conserved in RumA (8).…”

Section: Contacts With the Consensus Fold Are Highly Conserved Betweementioning

confidence: 99%

Section: Contacts With the Consensus Fold Are Highly Conserved Betweementioning

confidence: 99%

See 3 more Smart Citations

Structure of a TrmA–RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m ⁵ U methyltransferases

Alian¹,

Lee²,

Griner³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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100

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Base Flipping

Cheng¹,

Roberts²

2010

Encyclopedia of Life Sciences

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Base flipping involves the rotation of backbone bonds in double‐stranded deoxyribonucleic acid (DNA) to expose an out‐of‐stack base, which can then be a substrate for an enzyme‐catalysed chemical reaction or for a specific protein‐binding interaction. The phenomenon is fully established structurally and experimentally for DNA methyltransferases , for several key DNA repair enzymes, and for a few nonenzymatic DNA‐binding proteins involved in the DNA methylation and repair pathways, and is likely to prove general for enzymes or proteins that require access to unpaired, mismatched, damaged or modified bases. Recent results suggest that it may also be involved in the initial opening of a DNA helix during the initiation of transcription. Key Concept: Base flipping is a key mechanism used by many DNA and RNA modifying enzymes, including DNA repair enzymes, to gain access to one or more bases in a double helix.

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Base Flipping

Cheng

Roberts

2014

Encyclopedia of Life Sciences

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Base flipping involves rotation of backbone bonds in double‐stranded deoxyribonucleic acid (DNA) to expose an out‐of‐stack base, which can then be a substrate for an enzyme‐catalysed chemical reaction or for a specific protein binding interaction. The phenomenon is fully established for DNA methyltransferases, for several key DNA repair enzymes, and for a few non‐enzymatic DNA binding proteins involved in the DNA methylation and repair pathways, and is likely to prove general for enzymes or proteins that require access to unpaired, mismatched, damaged or modified bases or even undamaged and unmodified bases for specific functions. Although, detailed mechanistic information is emerging, it remains to be proven for each individual system whether base flipping is an active process in which the protein rotates the base out of the helix, or a passive one in which the protein binds to a transiently flipped base. Key Concepts: Base flipping is a key mechanism used by DNA and RNA modifying enzymes including DNA repair enzymes. DNA and RNA methyltransferases use AdoMet as the methyl donor to methylate flipped nucleotides (cytosine or adenine). The family of Fe(II)‐ and α‐ketoglutarate‐dependent dioxygenases includes members of the Tet family and AlkB‐like DNA/RNA repair enzymes. To date, six structural superfamilies of DNA repair glycosylases and newly identified (non‐repair) sequence‐specific DNA glycosylases all use base‐flipping mechanism to access the target base. Eukaryotic SRA domains share structural similarity to that of bacterial modification‐specific endonucleases (MspJI and AbaSI) in recognition of modified bases. Base flipping provides a simple mechanism by which a DNA helix can be opened during the initiation of polymerisation by DNA or RNA polymerases and their associated factors.

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A Unique RNA Fold in the RumA-RNA-Cofactor Ternary Complex Contributes to Substrate Selectivity and Enzymatic Function

Cited by 100 publications

References 47 publications

Structure of a TrmA–RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m ⁵ U methyltransferases

Structure of a TrmA–RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m ⁵ U methyltransferases

Base Flipping

Base Flipping

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A Unique RNA Fold in the RumA-RNA-Cofactor Ternary Complex Contributes to Substrate Selectivity and Enzymatic Function

Cited by 100 publications

References 47 publications

Structure of a TrmA–RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m 5 U methyltransferases

Structure of a TrmA–RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m 5 U methyltransferases

Base Flipping

Base Flipping

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Resources

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Structure of a TrmA–RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m ⁵ U methyltransferases

Structure of a TrmA–RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m ⁵ U methyltransferases