Ribosomes form the core of the protein biosynthesis machinery and are essential to life. Ribosome biogenesis is a complex cellular process involving transcription of rRNA, pre-rRNA processing, rRNA modification and simultaneous assembly of ribosomal proteins. RNA nucleotide modification is observed in all domains of life. While there is enormous conservation of ribosome structure, very few post-transcriptional rRNA modifications have been conserved throughout evolution. A notable example of such rare conservation is the dimethylation of two adjacent adenosines in the 3’-terminal helix, a highly conserved region of the small subunit rRNA. Enzymes that carry out these dimethylations are equally conserved and are collectively known as the KsgA/Dim1 family of methyltransferases. The first member of the family, KsgA, was identified in E. coli as the determinant for resistance to the aminoglycoside antibiotic Kasugamycin. Orthologs have since been described in organisms of wide spread evolutionary origins as well as in eukaryotic cellular organelles, thus underscoring the unprecedented conservation of this family of enzymes and the resultant rRNA modification. The higher evolutionary orthologs of KsgA have adopted secondary roles in ribosome biogenesis in addition to their dimethyltransferase role. The eukaryotic ortholog, Dim1, is essential for proper processing of the primary rRNA transcript. Recently, KsgA has been speculated to function as a late stage ribosome biogenesis factor and a ΔksgA genotype in E. coli has been linked to cold sensitivity and altered ribosomal profiles. This report focuses on the biochemical characterization of KsgA and its interaction with the 30S subunit. We have established the salt conditions required for optimal KsgA methyltransferase activity while confirming that KsgA recognizes a translationally inactive conformation of 30S subunit in vitro. Our study of the functional conservation of KsgA/Dim1 enzymes in the bacterial system revealed that KsgA and the evolutionarily higher orthologs could recognize a common ribosomal substrate. This indicates that the recognition elements of both, the protein and the small subunit, have remained largely unchanged during the course of evolution. Finally, based on our site directed mutagenesis and biochemical studies, we report that KsgA binds to structural components of 16S rRNA other than the helix containing the target nucleosides.

Eukaryotic ribosome biogenesis, a dynamic and coordinated multistep process which requires more than 150 trans-acting factors, has been intensely studied in the yeast Saccharomyces cerevisiae. This evolutionarily conserved process involves numerous cleavages of pre-rRNA, modification of nucleotides, and concomitant assembly of the ribosomal proteins onto the rRNA. Considerable information is available about the importance of conserved pre-rRNA cleavage events in ribosome biogenesis; however, very little is known about the exact role of modified nucleotides, which cluster within the functionally important regions of the ribosome. One conserved group of modifications is the dimethylation of two adjacent adenosines at the 3´ end of the small subunit rRNA which is ubiquitously carried out by the Dim1/KsgA methyltransferase family. Although dimethylation and KsgA are dispensable for survival in bacteria, the eukaryotic enzyme Dim1 is essential because of its requirement in the early pre-rRNA processing events. Similarly, few other members of the family have also evolved to carryout a second unrelated function in the cell. Almost all of the information about Dim1 was obtained from in vivo experiments in yeast, and has been determined that it is an indispensable part of a RNA-protein complex carrying out the pre-rRNA processing. Sequence analysis clearly shows that eukaryotic and archaeal enzymes have an extra insert in their C-terminal domain which is absent in bacterial enzymes and a better understanding of Dim1's function is only possible by its structural characterization which is the aim of this study. After several attempts, the yeast Dim1p was expressed under mild conditions in E. coli and purified in soluble form. Dim1p was able to methylate bacterial 30S subunits both in vivo and in vitro, indicating its ability to recognize bacterial substrate. Supporting our hypothesis, neither the bacterial nor archaeal orthologs were able to complement the processing function of Dim1p in yeast, tested using the plasmid shuffling technique. Our results suggest that the C-terminal insert of Dim1p, along with some structural features of the N-terminal domain, is important for its function in pre-rRNA processing. Further studies are required to understand the complex interactions between proteins and RNA involved in the ribosome biogenesis.

Antibiotic resistance threatens to throw mankind back into an era when infectious disease was the predominant cause of death. In an effort to mitigate this danger, we targeted ErmC’ and KsgA. Both methylate N6-adenosine of ribosomal RNA, though each serve different roles in their bacterial host. ErmC’ dimethylates A2058 on 23S rRNA, conferring resistance to MLSB antibiotics (macrolides, lincosamides, streptogramin B). KsgA aids in ribosome assembly, binding inactive 30S until dimethylating A1518/A1519 of 16S rRNA. Like most methyltransferases, ErmC’ and KsgA use cofactor S-adenosylmethionine (SAM) as their methyl source, which binds adjacent to their specific adenosine substrate. ErmC’ inhibitors could restore MLSB antibiotics against infections with this resistance mechanism. KsgA inhibitors could form novel antibiotics that stall 30S assembly. Previous studies reported a potent ErmC’ inhibitor, N6-cyclopentyl adenosine (1), binding to the substrate pocket with cyclopentyl bridging into the SAM pocket. We expanded this study by synthesizing 1 and 22 other N6-substituted analogs to explore more favorable interactions within the SAM pocket. When these compounds (1mM) were screened against ErmC’ and KsgA, some showed greater inhibition than 1. Two of these inhibitors that were crystallized with ErmC’, N6-8-octylamine adenosine (2.60Å) and N6-phenethyl adenosine (2.40Å), unexpectedly docked into the SAM pocket with their 5’-C pointing towards the substrate pocket. New compounds were made to exploit this orientation by adding substituents off the 5’-C to probe the substrate pocket. Through a five step synthesis, the 5’-OH of 1 was substituted with an amine linked to benzyl (30), phenethyl (31), propylphenyl (32) or butylphenyl (33). When 30-33 were screened using 20μM SAM, ErmC’ showed greater inhibition (relative to 1), while KsgA showed virtually none. However, when ErmC’ was tested using 0.5μM SAM, inhibition from 30-33 was nearly unchanged, whereas 1 became significantly more potent than 30-33, suggesting 30-33 were not binding to the SAM pocket. Preliminary data showed that raising 23S concentrations lowered inhibition from 32-33, while inhibition from 1, 30 and 31 was nearly unchanged, suggesting that at least 32-33 bound within the substrate pocket.