Substrate specificity and properties of the Escherichia coli 16S rRNA methyltransferase, RsmE

Abstract

The small ribosome subunit of Escherichia coli contains 10 base-methylated sites distributed in important functional regions. At present, seven enzymes responsible for methylation of eight bases are known, but most of them have not been well characterized. One of these enzymes, RsmE, was recently identified and shown to specifically methylate U1498. Here we describe the enzymatic properties and substrate specificity of RsmE. The enzyme forms dimers in solution and is most active in the presence of 10-15 mM Mg(2+) and 100 mM NH(4)Cl at pH 7-9; however, in the presence of spermidine, Mg(2+) is not required for activity. While small ribosome subunits obtained from an RsmE deletion strain can be methylated by purified RsmE, neither 70S ribosomes nor 50S subunits are active. Likewise, 16S rRNA obtained from the mutant strain, synthetic 16S rRNA, and 3' minor domain RNA are all very poor or inactive as substrates. 30S particles partially depleted of proteins by treatment with high concentrations of LiCl or in vitro reconstituted intermediate particles also show little or no methyl acceptor activity. Based on these data, we conclude that RsmE requires a highly structured ribonucleoprotein particle as a substrate for methylation, and that methylation events in the 3' minor domain of 16S rRNA probably occur late during 30S ribosome assembly.

FIGURE 1.

Primer extension analysis of total cellular RNA. Extracted RNA was annealed to a [³²P]-labeled primer complementary to residues 1506–1525 in 16S rRNA, prior to extension by M-MLV-RT as described in Materials and Methods. Products of the reaction were separated on 8% polyacrylamide/7 M urea denaturing gels and visualized by autoradiography. A stop at position A1499, noted by the arrow, indicates the presence of a methyl group at U1498. Data similar to those in lanes 1–5 were reported previously (Basturea et al. 2006). They were repeated for this experiment and are included as controls.

FIGURE 2.

Effect of Mg²⁺ and spermidine on RsmE activity. Ribosome particles (0.5 μM) were methylated with [³H]-SAM (100 μM) and purified His-RsmE (165 nM), as described in Materials and Methods. The amount of [³H]-methyl groups incorporated was monitored by acid precipitation and scintillation counting. One experiment representative of three repeats is shown. Although optima were identical among the three experiments, absolute incorporation varied due to use of different ribosome preparations.

FIGURE 3.

RsmE activity using ribosomal particles and various RNAs as substrates. (A) Activity on ribosome particles. 30S (■), 70S (●), or 50S (▲) ribosomes at 1 μM were methylated with [³H]-SAM (100 μM) and purified His-RsmE (165 nM) as described in Materials and Methods. (B) Activity on free RNAs. 30S subunits (■), 16S RNA (●), or total cellular RNA extracted from the rsmE deletion strain (▲), synthetic 16S RNA (○), or 3′ minor domain RNA (◆) at 1 μM were methylated under the same conditions as in A. The amount of [³H]-methyl groups incorporated was monitored by acid precipitation and scintillation counting. One experiment representative of three repeats is shown. Although relative incorporation was identical among the three experiments, absolute incorporation varied with the use of different ribosome preparations.

FIGURE 4.

RsmE activity on LiCl-washed ribosomal particles. (A) Sucrose gradient profiles of LiCl-washed particles. Mutant 30S particles were either mock-treated (■) or treated with 1 M LiCl (□), 1.5 M LiCl (●), 2 M LiCl (○), 3 M LiCl (▲), or 3.5 M LiCl (Δ) as described in Materials and Methods, and analyzed on sucrose gradients (top to the right). (B) Activity on LiCl-washed ribosomal particles. 16S RNA, intact 30S subunits, or 30S particles (2 μM) washed with the indicated concentrations of LiCl were methylated with [³H]-SAM (100 μM) and purified His-RsmE (165 nM) as described in Materials and Methods. The amount of [³H]-methyl groups incorporated was monitored by acid precipitation and scintillation counting. Data presented are the average of two experiments.

FIGURE 5.

RsmE activity of in vitro reconstituted intermediate particles. (A) Sucrose gradient profile of the in vitro reconstitution intermediate (●) (top to the right). In vitro reconstitution reactions were performed as described in Materials and Methods. ³²P-labeled ribosomal RNAs extracted from 70S ribosomes were used as markers (○). (B) Activity of the reconstitution intermediate. 16S RNA, purified natural 30S subunit, or 21S reconstitution intermediate (RI) (0.5 μM) were methylated with [³H]-SAM (100 μM) and purified His-tagged RsmE (165 nM) as described in Materials and Methods. The amount of [³H]-methyl groups incorporated was monitored by acid precipitation and scintillation counting. Data presented are the average of three experiments.