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Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. (Selmer)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. (Forster)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. (Forster)
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
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2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 20, 10507-20 p.Article in journal (Refereed) Published
Abstract [en]

RlmM (YgdE) catalyzes the S-adenosyl methionine (AdoMet)-dependent 2'O methylation of C2498 in 23S ribosomal RNA (rRNA) of Escherichia coli. Previous experiments have shown that RlmM is active on 23S rRNA from an RlmM knockout strain but not on mature 50S subunits from the same strain. Here, we demonstrate RlmM methyltransferase (MTase) activity on in vitro transcribed 23S rRNA and its domain V. We have solved crystal structures of E. coli RlmM at 1.9 Å resolution and of an RlmM-AdoMet complex at 2.6 Å resolution. RlmM consists of an N-terminal THUMP domain and a C-terminal catalytic Rossmann-like fold MTase domain in a novel arrangement. The catalytic domain of RlmM is closely related to YiiB, TlyA and fibrillarins, with the second K of the catalytic tetrad KDKE shifted by two residues at the C-terminal end of a beta strand compared with most 2'O MTases. The AdoMet-binding site is open and shallow, suggesting that RNA substrate binding may be required to form a conformation needed for catalysis. A continuous surface of conserved positive charge indicates that RlmM uses one side of the two domains and the inter-domain linker to recognize its RNA substrate.

Place, publisher, year, edition, pages
2012. Vol. 40, no 20, 10507-20 p.
National Category
Structural Biology
Identifiers
URN: urn:nbn:se:uu:diva-187880DOI: 10.1093/nar/gks727ISI: 000310970700054PubMedID: 22923526OAI: oai:DiVA.org:uu-187880DiVA: diva2:575820
Available from: 2012-12-11 Created: 2012-12-11 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Ribosomal RNA Modification Enzymes: Structural and functional studies of two methyltransferases for 23S rRNA modification in Escherichia coli
Open this publication in new window or tab >>Ribosomal RNA Modification Enzymes: Structural and functional studies of two methyltransferases for 23S rRNA modification in Escherichia coli
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Escherichia coli ribosomal RNA (rRNA) is post-transcriptionally modified by site-specific enzymes. The role of most modifications is not known and little is known about how these enzymes recognize their target substrates. In this thesis, we have structurally and functionally characterized two S-adenosyl-methionine (SAM) dependent 23S rRNA methyltransferases (MTases) that act during the early stages of ribosome assembly in E. coli.

RlmM methylates the 2'O-ribose of C2498 in 23S rRNA. We have solved crystal structures of apo RlmM at 1.9Å resolution and of an RlmM-SAM complex at 2.6Å resolution. The RlmM structure revealed an N-terminal THUMP domain and a C-terminal catalytic Rossmann-fold MTase domain. A continuous patch of conserved positive charge on the RlmM surface is likely used for RNA substrate recognition. The SAM-binding site is open and shallow, suggesting that the RNA substrate may be required for tight cofactor binding. Further, we have shown RlmM MTase activity on in vitro transcribed 23S rRNA and its domain V.

RlmJ methylates the exocyclic N6 atom of A2030 in 23S rRNA. The 1.85Å crystal structure of RlmJ revealed a Rossmann-fold MTase domain with an inserted small subdomain unique to the RlmJ family. The 1.95Å structure of the RlmJ-SAH-AMP complex revealed that ligand binding induces structural rearrangements in the four loop regions surrounding the active site. The active site of RlmJ is similar to N6-adenine DNA MTases. We have shown RlmJ MTase activity on in vitro transcribed 23S rRNA and a minimal substrate corresponding to helix 72, specific for adenosine. Mutagenesis experiments show that residues Y4, H6, K18 and D164 are critical for catalytic activity.

These findings have furthered our understanding of the structure, evolution, substrate recognition and mechanism of rRNA MTases.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1107
Keyword
Escherichia coli, ribosome biogenesis, ribosome assembly, ribosomal RNA, peptidyltransferase center, domain V, post-transcriptional modification, methyltransferases, S-adenosyl-methionine, RlmM, Cm2498, RlmJ, m6A2030, X-ray crystallography, substrate recognition, substrate specificity, catalytic mechanism, evolution
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biology with specialization in Structural Biology; Biochemistry
Identifiers
urn:nbn:se:uu:diva-212394 (URN)978-91-554-8834-5 (ISBN)
Public defence
2014-02-14, B42, Biomedical Center, Husargatan 3, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2014-01-22 Created: 2013-12-10 Last updated: 2014-02-10

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