Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Purification, crystallization and preliminary X-ray diffraction analysis of the 23S rRNA methyltransferase RlmJ from Escherichia coli
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
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).

Place, publisher, year, edition, pages
2013. Vol. 69, 1001-1003 p.
National Category
Structural Biology
Identifiers
URN: urn:nbn:se:uu:diva-208048DOI: 10.1107/S1744309113020289ISI: 000323719700010OAI: oai:DiVA.org:uu-208048DiVA: diva2:651115
Available from: 2013-09-24 Created: 2013-09-23 Last updated: 2017-12-06Bibliographically 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

Open Access in DiVA

fulltext(419 kB)105 downloads
File information
File name FULLTEXT01.pdfFile size 419 kBChecksum SHA-512
bce7782d7cbbea3a7ffcded97cb6925f8d101ac924daedbe23973e6376a17eccbbad1e6ba01e7fcd8c526456fcbaee9157112b111daa61fb260923a2ca57623a
Type fulltextMimetype application/pdf

Other links

Publisher's full text

Search in DiVA

By author/editor
Punekar, Avinash S.Selmer, Maria
By organisation
Structure and Molecular Biology
In the same journal
Acta Crystallographica. Section F: Structural Biology and Crystallization Communications
Structural Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 105 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 449 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf