Title

Identification and characterization of RsmE, the Escherichia coli methyltransferase responsible for methylation at U1498 in 16S rRNA

Date of Award

2007

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Murray P. Deutscher, Committee Chair

Abstract

A variety of RNA methyltransferases act during ribosomal RNA maturation to modify nucleotides in a site-specific manner. However, when this thesis was initiated, of the ten base-methylated nucleotides present in the small ribosomal subunit of E. coli, only three enzymes responsible for modification of four bases were known. In this work, I have shown that the protein encoded by rsmE, previously named yggJ , is responsible for methylation at U1498 in 16S rRNA. Thus, an rsmE deletion strain lacks the methyl group at U1498 as well as the specific methyltransferase activity. Interestingly, RsmE is the first member of a novel, distinct family of alpha/beta (trefoil) knot methyltransferases. While the deletion strain is essentially unaffected in exponential growth in rich or minimal media at multiple temperatures, it is defective when grown in competition with isogenic wild type cells. Purified RsmE specifically methylates m3U1498 in vitro. The enzyme forms dimers in solution and works best in the presence of 10--20 mM Mg2+ and 100 mM NH4Cl at pH 7--9. Although in the presence of spermidine, Mg2+ is not required for activity. Small ribosomal subunits, but neither 70S ribosomes nor 50S subunits, obtained from the rsmE deletion strain are methylated by purified RsmE. 16S rRNA obtained from the mutant strain is also a very poor substrate; as are synthetic 16S RNA and 3'-minor domain RNA. 30S particles partially depleted of proteins by treatment with high concentrations of LiCl or in vitro reconstituted intermediate particles show little or no methyl acceptor activity. Based on these data we conclude that the enzyme specifically methylates U1498 and requires a highly structured ribonucleoprotein particle as a substrate for catalysis, and that methylation events in the 3' minor domain of 16S rRNA probably occur late during 30S ribosome assembly.

Keywords

Biology, Molecular

Link to Full Text

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