The crystal structure of Emg1 revealed that it is a novel member of the alpha/beta knot fold MTase superfamily (). The alpha/beta knot fold in SCOP (26
) currently encompasses five families: (i) The YbeA-like family (pdb code 1ns5), with four uncharacterized members, which do not contain any specific additions to the core fold. (ii) The TrmD tRNA (m1G37)-MTases methylate the N1 position of guanine in tRNA. Their fold is similar to the YbeA-like family but contains an all-alpha C-terminal subdomain. (iii) The SpoU-like RNA 2′-O
ribose MTase family contains both tRNA (TrmH) and rRNA (RlmB) MTases, which methylate the ribose 2′-OH of guanosine at specific positions of tRNA or rRNA, respectively. RlmB contains an N-terminal domain with analogy to the L30 ribosomal protein linked to the catalytic subunit by a flexible linker. (iv) The archaeal Methanobacterium thermoautotrophicum
MT1 protein family, which is unique due to the insertion of an OB fold subdomain between β2 and β3. (v) The YggJ-like family that contains a N-terminal L5 ribosomal like domain. The core of Emg1 could be superimposed well with the core region of all members of the alpha/beta knot fold MTase superfamily. However, the Emg1 structure shows insertions that make it clearly distinct from each of these families. We therefore confirmed the prediction that Emg1 is the founding member of an additional alpha/beta knot fold MTase subfamily (15
The Emg1-specific insertions consist of two subdomains that form an extended surface on one side of the protein. This contributes to a basic patch around the active site, which is very likely involved in substrate rRNA binding (D), and Emg1 was indeed shown to bind RNA directly (C). Mutation of Arginine 88 (R88D), which is located within the basic patch, almost completely abolishes the interaction with the RNA, supporting the proposed function of this region in substrate binding. This conclusion is further supported by the finding of two sulfate ions in the structure that could mimic phosphates of the RNA backbone. The two sulfates are bound to the protein by salt bridges to Lys135/Arg136 and Arg136/Arg129. These three residues are located on the Emg1-specific region, αc′ helix and αc′-β3 loop and Arg136/Arg129 are absolutely conserved in both Eukaryotes and Archaea. This strongly suggests that this extended Emg1-specific region is also involved in RNA binding.
The results from the RNA-binding experiments are consistent with the findings of Buchhaupt et al. (2006), where a modified yeast three-hybrid approach was performed to identify a sequence derived from the 18S rRNA that was bound by Emg1 with high affinity. Our in vitro analyses showed that Emg1 binds to this 18S sequence more strongly than an RNA of unrelated sequence. However, in vitro binding was clearly not highly specific.
There is a striking difference between the sequence conservation of Emg1 in Eukaryotes (on average 50% identical) versus that in Archaea (on average 30% identical) (). The alignment suggests that the archaeal and eukaryotic Emg1 orthologs have the same characteristic fold but constitute two separate families, which might indicate that Emg1 has overlapping but not identical functions in both kingdoms of life. Surface residues conserved between Eukaryotes and Archaea cluster on one side of the protein: the SAM-binding site, the region immediately proximate to the methyl donor, the αA and αE face of the protein and the Emg1-specific extensions (C, left panel). In contrast, residues on the opposite side of the protein (Helix HC side, C right panel) are much less conserved between Eukaryotes and Archaea. While the αA and αE conserved face of Emg1 is likely involved in dimerization and/or substrate binding, the opposite, less conserved face, may interact with additional factors that are not conserved between Eukaryotes and Archaea. A candidate for such a factor could be Nop14, which binds to Emg1 in yeast (10
) but has no clear archaeal homolog.
A feature shared between all previously characterized members of the alpha/beta knot MTase families is the presence of dimers in the crystal forms, and some members were confirmed to form dimers in solution (27
). The detailed dimerization mode varies, but always involves the αA and αE helical surface of the protein. Moreover, mutants of the SpoU family protein Yibk, engineered to be monomeric rather than dimeric, were unable to bind SAH, suggesting that dimerization is required for structure and function of alpha-beta knot MTase (28
). In contrast, Emg1 crystallized as a monomer, possibly due to differences in the dimerization interface, the composition of the active site or disruption of the dimer by the crystallization liquor. Stable dimerization was, however, confirmed for Emg1 in solution, both by gel filtration and binding experiments ().
Based on the structure of the SAM-binding region of Emg1, site-directed mutagenesis was performed to obtain mutants defective in SAM binding. All mutants analyzed were found to be defective in binding SAM in vitro (), confirming predictions based on the structural data. The almost complete loss of affinity for SAM, the MTase cofactor, strongly indicates that these mutants have lost the capacity to perform a MTase reaction.
Emg1 is essential for viability in yeast, and we assessed whether the essential function requires its MTase activity or RNA binding. Three emg1 mutants that had lost SAM binding as well as the R88D mutant that lacks RNA-binding activity were able to fully complement the growth defect seen upon depletion of endogenous Emg1, suggesting that the presence of the catalytically inactive protein was sufficient to support growth (). Analysis of ribosome biogenesis in the SAM-binding mutants revealed that expression of only the inactive forms of Emg1 also supported normal pre-rRNA processing and ribosome synthesis (). We conclude that the putative SAM-dependant MTase activity of Emg1 is not required for ribosome biogenesis or growth in yeast.
Similar results were previously obtained for the MTase Dim1 and for methylation guide snoRNPs. Dim1 dimethylates two adjacent adenosine residues in the 18S rRNA (9
). Analyses of Dim1 showed that, as for Emg1, the presence of catalytically inactive protein was sufficient for ribosome biogenesis (9
). This leads to two possible interpretations. Both MTases may play structural roles in the pre-ribosome that are distinct from their enzymatic activities. Alternatively, or in addition, quality control mechanisms may have evolved to check for the presence of the protein rather than for the successful modification of the rRNA. Methylation guide snoRNPs carry out 2′-OH methylation at several sites on the rRNA. Strains lacking the activity of any individual methylation guide snoRNA were also viable (29
), whereas loss of the activity of the snoRNA-associated MTase Nop1, was lethal (30
). These data reveal that loss of rRNA methylation at any single site tested has little effect on ribosome biogenesis or function in yeast, whereas loss of methylation at multiple sites impairs ribosome synthesis and/or translation.