Ribosomal RNA Modification Enzymes : Structural and functional studies of two methyltransferases for 23S rRNA modification in Escherichia coli

• 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.

Subject headings

NATURVETENSKAP  -- Biologi -- Strukturbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Structural Biology (hsv//eng)

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

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

Biology with specialization in Structural Biology

Biologi med inriktning mot strukturbiologi

Biokemi

Biochemistry

Publication and Content Type

vet (subject category)

dok (subject category)