Structure And Function Study Of The Streptococcus Pneumoniae Ribosomal RNA Methyltransferase RlmCD

In prokaryotes, there are a variety of post-translational modifications on mature ribosomal RNA, of which methylation is one of the most important modifications.Until now, many nucleotide modifications have been identified on the ribosome 16S small subunit and 23S large subunit. More than 1/3 of these nucleotides are clustered to the ribosome function area, for instance the peptidyl transferase center (PTC) and the nascent peptide exit tunnel (NEPT). Among them, domain IV and helix 35 of domain ? are two heavily modified regions, which are component of NEPT region.RNA methyltransferases often use the S-Adenosyl methionine (SAM) as a methyl donor and transfer the methyl group to various positions of nucleotide bases.All of the methyltransferases can be divided into five methyltransferase families according to the secondary structure topological folding of their methyl transfer centers, from Class I to Class V respectively. Class ? of the RFM family and Class IV of the SPOUT family is two largest families. The RFM family members form a classic Rossmann fold in its activity center and the SPOUT family members have similar sandwich conformation but characterized with a trefoil knot.spRlmCD is a Streptococcus pneumoniae methyltransferase which catalyzes U1939 of ribosomal 23S rRNA to m5U1939. In Escherichia coli, the spRlmCD homologue is ecRumA. Different to ecRumA, spRImCD has an additional m5U747 methyltransferase activity. In contrast, U747 methylation is catalyzed by another enzyme named ecRImC in Escherichia coli. In Bacillus subtilis, bsYefA is also homologous to spRlmCD and both of them have m5U747 and m5U1939 methyltransferase activities. The domain arrangement is similar in all of the three proteins, from the N terminal TRAM domain, central domain, to C terminal catalytic domain. SpRlmCD has an additional extended C terminal domain. Solved structure of ecRumA and U1939 RNA complex indicated that the groove between TRAM domain and catalytic domain of ecRumA stabilized the RNA 3' helix region, the central domain interacted with 5' single strand region of the RNA, and uridine 1939 inserted into the catalytic center. Methylation of U747 is necessary for further methylation of 6748 to m1G748. which is important for erythromycin resistance of gram-positive bacteria. To provide structural basis of why spRImCD can methylate both U747 and U1939 while ecRumA can only methylate U1939, we sought to determine the structure of spRlmCD.In this thesis study, high-resolution crystal structure of the apo-form spRlmCD was determined. Stuctural analysis of its catalytic domain indicated that it belongs to Class ? methyltransferase family. In addition, spRlmCD-SAM-U747 18mer RNA complex structure was determined, and SAM -binding pocket was identified. With the consideration of non-specific interactions between protein and RNA during crystal packing, the structures of spRlmCD and ecRumA were compared. It showed that spRlmCD had two more excessive linkers in its central domain compare with ecRumA. Sequence alignment shows that bsYefA and ecRlmC both have these two excessive linkers as well. We speculated that the linkers at central domain are necessary for spRlmCD for the m5U747 methyl transfer activity. In vitro methyltransferase activity assay confirmed this linker is essential for the enzyme activity of RlmCD.