Site-directed mutants of 16S rRNA reveal important RNA domains for KsgA function and 30S subunit assembly

Abstract

KsgA is an rRNA methyltransferase important to the process of small subunit biogenesis in bacteria. It is ubiquitously found in all life including archaea and eukarya, where the enzyme is referred to as Dim1. Despite the emergence of considerable data addressing KsgA function over the last several years, details pertaining to RNA recognition are limited, in part because the most accessible substrate for in vitro studies of KsgA is the 900000 Da 30S ribosomal subunit. To overcome challenges imposed by size and complexity, we adapted recently reported techniques to construct in vivo assembled mutant 30S subunits suitable for use in in vitro methyltransferase assays. Using this approach, numerous 16S rRNA mutants were constructed and tested. Our observations indicate that the 790 loop of helix 24 plays an important role in overall catalysis by KsgA. Moreover, the length of helix 45 also is important to catalysis. In both cases loss of catalytic function occurred without an increase in the production of N(6)-methyladenosine, a likely indication that there was no critical reduction in binding strength. Both sets of observations support a "proximity" mechanism of KsgA function. We also report that several of the mutants constructed failed to assemble properly into 30S subunits, while some others did so with reduced efficiency. Therefore, the same technique of generating mutant 30S subunits can be used to study ribosome biogenesis on the whole.

Figure 1

Annotated 16S rRNA secondary structure from E. coli. Boxed regions are segments predicted to interact with KsgA in the context of a fully assembled 30S subunit. The figure of 16S rRNA is adapted from the work of Gutell, R. R. (35).

Figure 2

Sucrose density gradient profiles and in vitro time course KsgA methylation assay of 30S subunit substrates. (A) Sucrose gradient profile of 30S subunits isolated from MRE600 E. coli strain made kasugamycin resistant (30S^UnTag,UM) (B) Sucrose gradient analysis to determine the structural integrity of 30S subunits isolated from kasugamycin resistant DH5α strain expressing MS2 tagged 16S rRNA (30S^Tag,UM). (C) The three curves are time courses of KsgA transferring tritiated methyl groups from S-adenosyl-L-methionine to three types of 30S subunits. Blue Diamonds: 30S^UnTag,UM. Red Squares: 30S^Tag,UM. Green Triangles: Same as Red Squares, but from a strain sensitive to kasugamycin, therefore natively methylated at A1518 and A1519 (30S^Tag).

Figure 3

Disruption of sheared base pairings in helix 44 and KsgA activity. (A) The A1418C and A1483C mutations in helix 44 were constructed and tested for substrate activity. (B) and (C), respectively, represent the sucrose gradient profiles of A1418C and A1483C mutants. (D) Time course activity assays for KsgA catalyzed transfer of methyl groups to 30S^Tag,UM subunits (red) and the mutant subunits, 30S^Tag,UM[A1418C] (green) and 30S^Tag,UM[A1483C] (blue).

Figure 4

HPLC nucleotide analysis of KsgA methylated substrates mutated at A1418 and A1483. (A), (C), and (E), respectively, represent the traces of 30S^Tag,UM, 30S^Tag,UM[A1418C] and 30S^Tag,UM[A1483C] reactions at the 2 minute time point. (B), (D), and (F) represent products involving the same substrates, but at the end of 2 hr. The top trace in each panel is measured absorbance at 254 nm of a collection of nucleoside standards including m⁶A and m⁶₂A, while the bottom trace in each panel is measured ³H levels.

Figure 5

Effects of helix 24 loop mutations on 30S assembly efficiency and methylation by KsgA. (A) The loop nucleotides (787-795) were replaced with GAAA, a member of the GNRA tetraloop family. G791 was mutated to each of A, C, and U. (B) and (C), respectively, show the sucrose gradient analysis for the 790-loop⁻ mutant and the G791U mutant, which is representative of all three single G791 mutations. For G791 mutants, material corresponding to the 30S peak (peak 4 in G791U profile) was used for KsgA time course activity assays, while sucrose gradient fractions of earlier peaks (peaks 1, 2, and 3) were used in a 2-hr end point methylation assay. Fractions pooled for each peak are indicated by arrows matching in color to the peak number. (D) Time course activity assays for 30S^Tag,UM subunits (red) and the three mutant subunits, 30S^Tag,UM[G791A] (green), 30S^Tag,UM[G791C] (blue), and 30S^Tag,UM[G791U] (purple). (E) 2-hr end point methylation assay of sucrose gradient fractions of 30S^Tag,UM (red), 30S^Tag,UM[G791A] (green), 30S^Tag,UM[G791C] (blue), and 30S^Tag,UM[G791U] (purple).

Figure 6

HPLC nucleoside analysis of KsgA methylated substrates mutated in the loop of helix 24. (A), (B), and (C), respectively, are chromatograms involving nucleosides derived from 30S^Tag,UM[G791A], 30S^Tag,UM[G791C], and 30S^Tag,UM[G791U] substrates of the 2-hr end point reaction. The top trace in each panel is the measured absorbance at 254 nm of a collection of nucleoside standards including m⁶A and m⁶₂A, while the bottom trace in each panel is measured ³H levels.

Figure 7

Effects of lengthening helix 45 by two base pairs on methylation by KsgA. (A) A mutant composed of the addition of two base pairs in helix 45 was constructed and tested for substrate activity. (B) Sucrose gradient analysis for the add2bp mutant. Material corresponding to the 30S peak, indicated by arrows, was used for the activity assay. (C) KsgA time course activity assay of 30S^Tag,UM (red) and mutant 30S^Tag,UM[add2bp] (green) subunits. (D) Nucleoside analysis of the 2-hr end point reaction product. The top trace is measured absorbance at 254 nm of a collection of nucleoside standards including m⁶A and m⁶₂A, while the bottom trace is measured ³H levels.