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Crystal structure of RlmM, the 2'O-ribose methyltransferase for C2498 of Escherichia coli 23S rRNA
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. (Selmer)
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. (Forster)
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi. (Forster)
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Biologiska sektionen, Institutionen för cell- och molekylärbiologi, Struktur- och molekylärbiologi.
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2012 (Engelska)Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, nr 20, s. 10507-20Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
2012. Vol. 40, nr 20, s. 10507-20
Nationell ämneskategori
Strukturbiologi
Identifikatorer
URN: urn:nbn:se:uu:diva-187880DOI: 10.1093/nar/gks727ISI: 000310970700054PubMedID: 22923526OAI: oai:DiVA.org:uu-187880DiVA, id: diva2:575820
Tillgänglig från: 2012-12-11 Skapad: 2012-12-11 Senast uppdaterad: 2019-01-25Bibliografiskt granskad
Ingår i avhandling
1. Ribosomal RNA Modification Enzymes: Structural and functional studies of two methyltransferases for 23S rRNA modification in Escherichia coli
Öppna denna publikation i ny flik eller fönster >>Ribosomal RNA Modification Enzymes: Structural and functional studies of two methyltransferases for 23S rRNA modification in Escherichia coli
2014 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2014. s. 65
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1107
Nyckelord
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
Nationell ämneskategori
Strukturbiologi Biokemi och molekylärbiologi
Forskningsämne
Biologi med inriktning mot strukturbiologi; Biokemi
Identifikatorer
urn:nbn:se:uu:diva-212394 (URN)978-91-554-8834-5 (ISBN)
Disputation
2014-02-14, B42, Biomedical Center, Husargatan 3, Uppsala, 13:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2014-01-22 Skapad: 2013-12-10 Senast uppdaterad: 2014-02-10
2. Exotic Ribosomal Enzymology
Öppna denna publikation i ny flik eller fönster >>Exotic Ribosomal Enzymology
2019 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

This thesis clarifies intriguing enzymology of the ribosome, the multiRNA/multiprotein complex that catalyzes protein synthesis (translation). The large ribosomal RNAs (23S and 16S rRNAs in E. coli) are post-transcriptionally modified by many specific modification enzymes, yet the functions of the modifications remain enigmatic. A deeper insight into two of the 23S rRNA S-adenosyl-methionine-requiring methyltransferase enzymes, RlmM and RlmJ, was given by investigating substrate specificity in vitro. Both enzymes were able to methylate in vitro-transcribed, modification-free, protein-free, 2659-nucleotide-long 23S rRNA. Furthermore, RlmM was able to methylate the 611-nucleotide-long Domain V of the 23S rRNA alone and RlmJ could modify the A2030 with only 25 surrounding nucleotides.

Translation is evolutionary optimized to incorporate L-amino acids to the exclusion of D-amino acids in the cell. To understand how, and how to engineer around this restriction for pharmacological applications, detailed kinetics of ribosomal dipeptide formation with D- versus L-phenylalanine-tRNA were determined. This was done by varying the concentrations of EF-Tu (which delivers aminoacyl-tRNAs to the ribosome) and the ribosome, as well as changing the tRNA adaptor. Binding to EF-Tu was shown to be rate limiting for D-Phe-tRNA at a low concentration of EF-Tu. Surprisingly, at a higher (physiological) concentration of EF-Tu, binding and subsequent dipeptide synthesis became so efficient that D-Phe incorporation became competitive with L-Phe, and accommodation/peptide bond formation was unmasked as a new rate-limiting step. This highlighted the importance of D-aminoacyl-tRNA deacylase in restricting translation with D-amino acids in vivo.

Although polypeptides are intrinsically colorless, it is remarkable that evolution has nevertheless enabled ribosomes to synthesize highly colored proteins (chromoproteins). Such eukaryotic proteins reside in coral reefs and undergo self-catalyzed, intramolecular, chromophore formation by reacting with oxygen in a manner highly similar to that of green fluorescent protein. The potential utility of different colored chromoproteins in E. coli was analyzed via codon-optimized over-expression and quantification of maturation times, color intensities and cellular fitness costs. No chromoprotein was found to have the combined characteristics of fast maturation, intense color and low fitness cost. However, semi-rational mutagenesis created different colored variants with identical fitness costs suitable for competition assays and teaching.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2019. s. 43
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1770
Nyckelord
rRNA modification, Methyltransferase, RlmM, RlmJ, D-amino acid, Unnatural amino acid, Chromoprotein
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
Biologi med inriktning mot molekylärbiologi
Identifikatorer
urn:nbn:se:uu:diva-374965 (URN)978-91-513-0567-7 (ISBN)
Disputation
2019-03-08, A1:111a, BMC, Husargatan 3, Uppsala, 09:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2019-02-15 Skapad: 2019-01-25 Senast uppdaterad: 2019-03-18

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