Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function

Abstract

All organisms incorporate post-transcriptional modifications into ribosomal RNA, influencing ribosome assembly and function in ways that are poorly understood. The most highly conserved modification is the dimethylation of two adenosines near the 39 end of the small subunit rRNA. Lack of these methylations due to deficiency in the KsgA methyltransferase stimulates translational errors during both the initiation and elongation phases of protein synthesis and confers resistance to the antibiotic kasugamycin. Here, we present the X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit lacking these dimethylations. Our data indicate that the KsgA-directed methylations facilitate structural rearrangements in order to establish a functionally optimum subunit conformation during the final stages of ribosome assembly.

Structural Insights into Methyltransferase KsgA Function in 30S Ribosomal Subunit Biogenesis

“…We reactivated the 30Si ribosomal subunit by incubation in a high magnesium buffer at 40°C. In the three-dimensional reconstruction, we observe a well defined density for helix 44 in a conformation similar to the crystal structure of translationally active 30S subunits, confirming the structural integrity of our 30Si preparation (3,26). Thus, the absence of a helix 44 density in the reconstructions of the inactive 30Si ribosomal subunit is due to a conformational change of helix 44 upon inactivation.…”

“…These structures show catalytically inactive complexes and are inconsistent with structural model based on footprinting data (19). Other structural data on this system include the x-ray crystal structure of a 30S ribosomal subunit lacking the methylations on A1518 and A1519 (26). However, this structure was obtained under high magnesium conditions under which the subunit cannot be modified by KsgA.…”

The central role of protein S12 in organizing the structure of the decoding site of the ribosome

“…This finding indicates that the structural changes resulting from SmD mutations are readily reversible. The ability of streptomycin to favor a near-native conformation of the decoding site of the SmD ribosome is in line with our own recent crystallographic data showing the drug to stabilize binding of nearcognate anticodon stem-loop analogs (ASL) by favoring a near-native conformation of the decoding site of wild-type ribosomes (Demirci et al 2010). The binding energy of streptomycin, which interacts with several structural elements of the 30S subunit, is apparently sufficient to overcome the less favorable P90 side-chain conformation resulting from its steric clash with h44.…”

Nuclear Ribosome Biogenesis Mediated by the DIM1A rRNA Dimethylase Is Required for Organized Root Growth and Epidermal Patterning in Arabidopsis

“…Whereas structural analyses of KsgA in bacteria indicate that the two adenosine dimethylations are essential for optimal conformation of the small ribosomal subunit for translation (Xu et al, 2008;Demirci et al, 2010), we cannot necessarily conclude that this structural change also occurs in the Arabidopsis ribosome. In addition, we are unable to ascertain whether the absence of these methylations alone causes the developmental defects observed in the dim1A mutant background or if lack of the methylations disrupts binding of a ribosomal protein, affecting ribosome function.…”