Sequence and structural evolution of the KsgA/Dim1 methyltransferase family

<p>Abstract</p> <p>Background</p> <p>One of the 60 or so genes conserved in all domains of life is the <it>ksgA/dim1 </it>orthologous group. Enzymes from this family perform the same post-transcriptional nucleotide modification in ribosome biogenesis, irrespective of organism. Despite this common function, divergence has enabled some family members to adopt new and sometimes radically different functions. For example, in <it>S. cerevisiae </it>Dim1 performs two distinct functions in ribosome biogenesis, while human mtTFB is not only an rRNA methyltransferase in the mitochondria but also a mitochondrial transcription factor. Thus, these proteins offer an unprecedented opportunity to study evolutionary aspects of structure/function relationships, especially with respect to our recently published work on the binding mode of a KsgA family member to its 30S subunit substrate. Here we compare and contrast KsgA orthologs from bacteria, eukaryotes, and mitochondria as well as the paralogous ErmC enzyme.</p> <p>Results</p> <p>By using structure and sequence comparisons in concert with a unified ribosome binding model, we have identified regions of the orthologs that are likely related to gains of function beyond the common methyltransferase function. There are core regions common to the entire enzyme class that are associated with ribosome binding, an event required in rRNA methylation activity, and regions that are conserved in subgroups that are presumably related to non-methyltransferase functions.</p> <p>Conclusion</p> <p>The ancient protein KsgA/Dim1 has adapted to cellular roles beyond that of merely an rRNA methyltransferase. These results provide a structural foundation for analysis of multiple aspects of ribosome biogenesis and mitochondrial transcription.</p

<it>Staphylococcus aureus</it> and <it>Escherichia coli</it> have disparate dependences on KsgA for growth and ribosome biogenesis

<p>Abstract</p> <p>Background</p> <p>The KsgA methyltransferase has been conserved throughout evolution, methylating two adenosines in the small subunit rRNA in all three domains of life as well as in eukaryotic organelles that contain ribosomes. Understanding of KsgA’s important role in ribosome biogenesis has been recently expanded in <it>Escherichia coli</it>; these studies help explain why KsgA is so highly conserved and also suggest KsgA’s potential as an antimicrobial drug target.</p> <p>Results</p> <p>We have analyzed KsgA’s contribution to ribosome biogenesis and cell growth in <it>Staphylococcus aureus</it>. We found that deletion of <it>ksgA</it> in <it>S. aureus</it> led to a cold-sensitive growth phenotype, although KsgA was not as critical for ribosome biogenesis as it was shown to be in <it>E. coli</it>. Additionally, the <it>ksgA</it> knockout strain showed an increased sensitivity to aminoglycoside antibiotics. Overexpression of a catalytically inactive KsgA mutant was deleterious in the knockout strain but not the wild-type strain; this negative phenotype disappeared at low temperature.</p> <p>Conclusions</p> <p>This work extends the study of KsgA, allowing comparison of this aspect of ribosome biogenesis between a Gram-negative and a Gram-positive organism. Our results in <it>S. aureus</it> are in contrast to results previously described in <it>E. coli,</it> where the catalytically inactive protein showed a negative phenotype in the presence or absence of endogenous KsgA.</p

The structure of a methylated tetraloop in 16S ribosomal RNA

AbstractBackground: Ribosomal RNAs contain many modified nucleotides. The functions of these nucleotides are poorly understood and few of them are strongly conserved. The final stem loop in 16S-like rRNAs is an exception in both regards. In both prokaryotes and eukaryotes, the tetranucleotide loop that caps the 3′-terminal stem contains two N6,N6-dimethyladenosine residues. The sequence and pattern of methylation are conserved within the loop, and there is evidence that these methylated nucleotides play an important role in subunit association and the initiation of protein synthesis. Because of the integral role that helix 45 plays in ribosome function, it is important to know what consequences these methylated nucleotides have on its structure.Results: We have solved the solution structure of a 14-nucleotide analog of the terminal stem loop of bacterial 16S rRNA, which contains N2-methylguanosine as well as two N6,N6-dimethyladenosines.Conclusions: The methylation of the 16S rRNA stem loop completely alters its conformation, which would otherwise be a GNRA tetraloop. It is likely that the conformation of this loop is crucial for its function, having implications for its interaction with ribosomal subunits and its role in the initiation of protein synthesis

Recognition of a complex substrate by the KsgA/Dim1 family of enzymes has been conserved throughout evolution

Ribosome biogenesis is a complicated process, involving numerous cleavage, base modification and assembly steps. All ribosomes share the same general architecture, with small and large subunits made up of roughly similar rRNA species and a variety of ribosomal proteins. However, the fundamental assembly process differs significantly between eukaryotes and eubacteria, not only in distribution and mechanism of modifications but also in organization of assembly steps. Despite these differences, members of the KsgA/Dim1 methyltransferase family and their resultant modification of small-subunit rRNA are found throughout evolution and therefore were present in the last common ancestor. In this paper we report that KsgA orthologs from archaeabacteria and eukaryotes are able to complement for KsgA function in bacteria, both in vivo and in vitro. This indicates that all of these enzymes can recognize a common ribosomal substrate, and that the recognition elements must be largely unchanged since the evolutionary split between the three domains of life

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+2 dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+2 (translationally inactive) and high Mg+2 (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit

Journal of Biomolecular NMR, 10 (1997) 255--262 255 *To whom correspondence should be addressed.

Introduction The rate at which individual DNA and RNA molecules move through solution, i.e. the translational self-diffusion rate, is of fundamental importance for many important aspects of nucleic acid biochemistry. Any process which changes the apparent hydrodynamic parameters of a nucleic acid, such as protein or ligand binding, drug intercalation, or bending, can produce a measurable change in this diffusion rate. The NMR pulsed field-gradient (PFG) spin-echo technique (Hahn, 1950; Stejskal and Tanner, 1965) has long been used to measure diffusion constants. Applications to biological systems include determination of the aggregation state of proteins (Altieri et al., 1995; Dingley et al., 1995), measurement of the bulk movement of hemoglobin in human erythrocytes (Kuchel and Chapman, 1991) and quantitation of processes such as amide proton exchange with water (Andrec and Prestegard, 1996). For NMR spectroscopists, it provides a simple, accurate method for measuring the diffusio

Control of Substrate Specificity by a Single Active Site Residue of the KsgA Methyltransferase

The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and <i>N</i>⁶-methyladenosine as substrates to catalyze formation of the final product <i>N</i>⁶,<i>N</i>⁶-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of <i>S</i>-adenosyl-l-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the <i>N</i>⁶-methyladenosine intermediate to produce <i>N</i>⁶,<i>N</i>⁶-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of <i>N</i>⁶-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA’s multiple catalytic steps

Control of Substrate Specificity by a Single Active Site Residue of the KsgA Methyltransferase

The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and <i>N</i>⁶-methyladenosine as substrates to catalyze formation of the final product <i>N</i>⁶,<i>N</i>⁶-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of <i>S</i>-adenosyl-l-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the <i>N</i>⁶-methyladenosine intermediate to produce <i>N</i>⁶,<i>N</i>⁶-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of <i>N</i>⁶-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA’s multiple catalytic steps