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Ribosomal RNA Modification Enzymes: Structural and functional studies of two methyltransferases for 23S rRNA modification in Escherichia coli
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. (Maria Selmer)ORCID iD: 0000-0002-6541-9529
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 [en]
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: urn:nbn:se:uu:diva-212394ISBN: 978-91-554-8834-5 (print)OAI: oai:DiVA.org:uu-212394DiVA: diva2:679377
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
List of papers
1. Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA
Open this publication in new window or tab >>Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA
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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.

National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-187880 (URN)10.1093/nar/gks727 (DOI)000310970700054 ()22923526 (PubMedID)
Available from: 2012-12-11 Created: 2012-12-11 Last updated: 2017-12-07Bibliographically approved
2. Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli
Open this publication in new window or tab >>Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli
2013 (English)In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 69, 1001-1003 p.Article in journal (Refereed) Published
Abstract [en]

Methyltransferase RlmJ uses the cofactor S-adenosylmethionine to methylate the exocyclic nitrogen N6 of nucleotide A2030 in 23S rRNA during ribosome assembly in Escherichia coli. RlmJ with a C-terminal hexahistidine tag was overexpressed in E. coli and purified as a monomer using Ni2+-affinity and size-exclusion chromatography. The recombinant RlmJ was crystallized using the sitting-drop vapour-diffusion method and a full data set was collected to 1.85 angstrom resolution from a single apo crystal. The crystals belonged to space group P2(1), with unit-cell parameters a = 46.9, b = 77.8, c = 82.5 angstrom, beta = 104 degrees. Data analysis suggested two molecules per asymmetric unit and a Matthews coefficient of 2.20 angstrom(3) Da(-1).

National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-208048 (URN)10.1107/S1744309113020289 (DOI)000323719700010 ()
Available from: 2013-09-24 Created: 2013-09-23 Last updated: 2017-12-06Bibliographically approved
3. Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ
Open this publication in new window or tab >>Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ
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2013 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, no 20, 9537-9548 p.Article in journal (Refereed) Published
Abstract [en]

RlmJ catalyzes the m(6)A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain. Binding of cofactor and substrate induces a large shift of the N-terminal motif X tail to make it cover the cofactor binding site and trigger active-site changes in motifs IV and VIII. Adenosine monophosphate binds in a partly accommodated state with the target N6 atom 7 Å away from the sulphur of AdoHcy. The active site of RlmJ with motif IV sequence 164DPPY167 is more similar to DNA m(6)A MTases than to RNA m(6)2A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI. RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate. RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.

National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-211566 (URN)10.1093/nar/gkt719 (DOI)000326746400036 ()23945937 (PubMedID)
Available from: 2013-11-26 Created: 2013-11-26 Last updated: 2017-12-06Bibliographically approved

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