Ribosomal RNA guanine-(N2)-methyltransferases and their targets

Five nearly universal methylated guanine-(N2) residues are present in bacterial rRNA in the ribosome. To date four out of five ribosomal RNA guanine-(N2)-methyltransferases are described. RsmC(YjjT) methylates G1207 of the 16S rRNA. RlmG(YgjO) and RlmL(YcbY) are responsible for the 23S rRNA m²G1835 and m²G2445 formation, correspondingly. RsmD(YhhF) is necessary for methylation of G966 residue of 16S rRNA. Structure of Escherichia coli RsmD(YhhF) methyltransferase and the structure of the Methanococcus jannaschii RsmC ortholog were determined. All ribosomal guanine-(N2)-methyltransferases have similar AdoMet-binding sites. In relation to the ribosomal substrate recognition, two enzymes that recognize assembled subunits are relatively small single domain proteins and two enzymes that recognize naked rRNA are larger proteins containing separate methyltransferase- and RNA-binding domains. The model for recognition of specific target nucleotide is proposed. The hypothetical role of the m²G residues in rRNA is discussed.     

Phylogenomic analysis of 16S rRNA:(guanine-N2) methyltransferases suggests new family members and reveals highly conserved motifs and a domain structure similar to other nucleic acid amino-methyltransferases.

The sequences of known Escherichia coli 16S rRNA:m2G1207 methyltransferase (MTase) RsmC and hypothetical 16S rRNA:m2G966 MTase encoded by the ygjo open reading frame were used to carry out a database search of other putative m2G-generating enzymes in finished and unfinished genomic sequences. Sequence comparison and phylogenetic analysis of 21 close homologs of RsmC and YgjO revealed the presence of the third paralogous lineage in E. coli and other gamma-Proteobacteria, which might correspond to the subfamily of MTases specific for G1516 in 16S rRNA. In addition, the comparative sequence analysis supported by sequence/structure threading suggests that rRNA:m2G MTases are very closely related to RNA and DNA:m6A MTases and that these two enzyme families share common architecture of the active site and presumably a similar mechanism of methyl group transfer onto the exocyclic amino group of their target bases.     

Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure

N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.     

Protein-independent folding pathway of the 16S rRNA 5' domain

Evolution of the ribosome from an RNA catalyst suggests that the intrinsic folding pathway of the rRNA dictates the hierarchy of ribosome assembly. To address this possibility, we probed the tertiary folding pathway of the 5' domain of the Escherichia coli 16S rRNA at 20 ms intervals using X-ray-dependent hydroxyl radical footprinting. Comparison with crystallographic structures and footprinting reactions on native 30S ribosomes showed that the RNA formed all of the predicted tertiary interactions in the absence of proteins. In 20 mM MgCl2, many tertiary interactions appeared within 20 ms. By contrast, interactions between H6, H15 and H17 near the spur of the 30S ribosome evolved over several minutes, likely due to mispairing of a central helix junction. The kinetic folding pathway of the RNA corresponded to the expected order of protein binding, suggesting that the RNA folding pathway forms the basis for early steps of ribosome assembly.     

The yeast ribosome synthesis factor Emg1 is a novel member of the superfamily of alpha/beta knot fold methyltransferases

Emg1 was previously shown to be required for maturation of the 18S rRNA and biogenesis of the 40S ribosomal subunit. Here we report the determination of the crystal structure of Emg1 at 2 Å resolution in complex with the methyl donor, S-adenosyl-methionine (SAM). This structure identifies Emg1 as a novel member of the alpha/beta knot fold methyltransferase (SPOUT) superfamily. In addition to the conserved SPOUT core, Emg1 has two unique domains that form an extended surface, which we predict to be involved in binding of RNA substrates. A point mutation within a basic patch on this surface almost completely abolished RNA binding in vitro. Three point mutations designed to disrupt the interaction of Emg1 with SAM each caused>100-fold reduction in SAM binding in vitro. Expression of only Emg1 with these mutations could support growth and apparently normal ribosome biogenesis in strains genetically depleted of Emg1. We conclude that the catalytic activity of Emg1 is not essential and that the presence of the protein is both necessary and sufficient for ribosome biogenesis.     

Structural insights into the function of 23S rRNA methyltransferase RlmG (m²G1835) from Escherichia coli

RlmG is a specific AdoMet-dependent methyltransferase (MTase) responsible for N2-methylation of G1835 in 23S rRNA of Escherichia coli. Methylation of m2G1835 specifically enhances association of ribosomal subunits and provides a significant advantage for bacteria in osmotic and oxidative stress. Here, the crystal structure of RlmG in complex with AdoMet and its structure in solution were determined. The structure of RlmG is similar to that of the MTase RsmC, consisting of two homologous domains: the N-terminal domain (NTD) in the recognition and binding of the substrate, and the C-terminal domain (CTD) in AdoMet-binding and the catalytic process. However, there are distinct positively charged protuberances and a distribution of conserved residues contributing to the charged surface patch, especially in the NTD of RlmG for direct binding of protein-free rRNA. The RNA-binding properties of the NTD and CTD characterized by both gel electrophoresis mobility shift assays and isothermal titration calorimetry showed that NTD could bind RNA independently and RNA binding was achieved by the NTD, accomplished by a coordinating role of the CTD. The model of the RlmG-AdoMet-RNA complex suggested that RlmG may unfold its substrate RNA in the positively charged cleft between the NTD and CTD, and then G1835 disengages from its Watson-Crick pairing with C1905 and flips out to insert into the active site. Our structure and biochemical studies provide novel insights into the catalytic mechanism of G1835 methylation.     

RNA:(guanine-N2) methyltransferases RsmC/RsmD and their homologs revisited – bioinformatic analysis and prediction of the active site based on the uncharacterized Mj0882 protein structure

Background  

Escherichia coli guanine-N2 (m²G) methyltransferases (MTases) RsmC and RsmD modify nucleosides G1207 and G966 of 16S rRNA. They possess a common MTase domain in the C-terminus and a variable region in the N-terminus. Their C-terminal domain is related to the YbiN family of hypothetical MTases, but nothing is known about the structure or function of the N-terminal domain.     

Leader peptides of inducible chloramphenicol resistance genes from gram-positive and gram-negative bacteria bind to yeast and Archaea large subunit rRNA.

catA86 is the second gene in a constitutively transcribed, two-gene operon cloned from Bacillus pumilus . The region that intervenes between the upstream gene, termed the leader, and the catA86 coding sequence contains a pair of inverted repeat sequences which cause sequestration of the catA86 ribosome binding site in mRNA secondary structure. As a consequence, the catA86 coding sequence is untranslatable in the absence of inducer. Translation of the catA86 coding sequence is induced by chloramphenicol in Gram-positives and induction requires a function of the leader coding sequence. The leader-encoded peptide has been proposed to instruct its translating ribosome to pause at leader codon 6, enabling chloramphenicol to stall the ribosome at that site. Ribosome stalling causes destabilization of the RNA secondary structure, exposing the catA86 ribosome binding site, allowing activation of its translation. A comparable mechanism of induction by chloramphenicol has been proposed for the regulated cmlA gene from Gram-negative bacteria. The catA86 and cmlA leader-encoded peptides are in vitro inhibitors of peptidyl transferase, which is thought to be the basis for selection of the site of ribosome stalling. Both leader-encoded peptides have been shown to alter the secondary structure of Escherichia coli 23S rRNA in vitro. All peptide-induced changes in rRNA conformation are within domains IV and V, which contains the peptidyl transferase center. Here we demonstrate that the leader peptides alter the conformation of domains IV and V of large subunit rRNA from yeast and a representative of the Archaea. The rRNA target for binding the leader peptides is therefore conserved across kingdoms.     

Sequence-structure-function analysis of the bifunctional enzyme MnmC that catalyses the last two steps in the biosynthesis of hypermodified nucleoside mnm5s2U in tRNA

MnmC catalyses the last two steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) in tRNA. Previously, we reported that this bifunctional enzyme is encoded by the yfcK open reading frame in the Escherichia coli K12 genome. However, the mechanism of its activity, in particular the potential structural and functional dependence of the domains responsible for catalyzing the two modification reactions, remains unknown. With the aid of the protein fold-recognition method, we constructed a structural model of MnmC in complex with the ligands and target nucleosides and studied the role of individual amino acids and entire domains by site-directed and deletion mutagenesis, respectively. We found out that the N-terminal domain contains residues responsible for binding of the S-adenosylmethionine cofactor and catalyzing the methylation of nm(5)s(2)U to form mnm(5)s(2)U, while the C-terminal domain contains residues responsible for binding of the FAD cofactor. Further, point mutants with compromised activity of either domain can complement each other to restore a fully functional enzyme. Thus, in the conserved fusion protein MnmC, the individual domains retain independence as enzymes. Interestingly, the N-terminal domain is capable of independent folding, while the isolated C-terminal domain is incapable of folding on its own, a situation similar to the one reported recently for the rRNA modification enzyme RsmC.     

A structural model for the assembly of the 30S subunit of the ribosome

The order in which proteins bind to 16S rRNA, the assembly map, was determined by Nomura and co-workers in the early 1970s. The assembly map shows the dependencies of binding of successive proteins but fails to address the relationship of these dependencies to the three-dimensional folding of the ribosome. Here, using molecular mechanics techniques, we rationalize the order of protein binding in terms of ribosomal folding. We determined the specific contacts between the ribosomal proteins and 16S rRNA from a crystal structure of the 30S subunit (1FJG). We then used these contacts as restraints in a rigid body Monte-Carlo simulation with reduced-representation models of the RNA and proteins. Proteins were added sequentially to the RNA in the order that they appear in the assembly map. Our results show that proteins nucleate the folding of the head, platform, and body domains, but they do not strongly restrict the orientations of the domains relative to one another. We also examined the contributions of individual proteins to the formation of binding sites for sequential proteins in the assembly process. Binding sites for the primary binding proteins are generally more ordered in the naked RNA than those for other proteins. Furthermore, we examined one pathway in the assembly map and found that the addition of early binding proteins helps to organize the RNA around the binding sites of proteins that bind later. It appears that the order of assembly depends on the degree of pre-organization of each protein's binding site at a given stage of assembly, and the impact that the binding of each protein has on the organization of the remaining unoccupied binding sites.     

Crystal structure of RumA, an iron-sulfur cluster containing E. coli ribosomal RNA 5-methyluridine methyltransferase

RumA catalyzes transfer of a methyl group from S-adenosylmethionine (SAM) specifically to uridine 1939 of 23S ribosomal RNA in Escherichia coli to yield 5-methyluridine. We determined the crystal structure of RumA at 1.95 A resolution. The protein is organized into three structural domains: The N-terminal domain contains sequence homology to the conserved TRAM motif and displays a five-stranded beta barrel architecture characteristic of an oligosaccharide/oligonucleotide binding fold. The central domain contains a [Fe(4)S(4)] cluster coordinated by four conserved cysteine residues. The C-terminal domain displays the typical SAM-dependent methyltransferase fold. The catalytic nucleophile Cys389 lies in a motif different from that in DNA 5-methylcytosine methyltransferases. The electrostatic potential surface reveals a predominately positively charged area that covers the concave surface of the first two domains and suggests an RNA binding mode. The iron-sulfur cluster may be involved in the correct folding of the protein or may have a role in RNA binding.     

Structure of the mouse nucleolin gene. The complete sequence reveals that each RNA binding domain is encoded by two independent exons

Nucleolin is a multifunctional nucleolar protein involved in the synthesis, packaging and maturation of pre-rRNA in eukaryotic cells. We describe the molecular organization and complete sequence of the mouse nucleolin gene, the first higher eukaryotic gene encoding a protein that is both an RNA binding protein involved in rRNA processing and a specific nucleolar protein. The nucleolin gene extends over 9000 base-pairs and is split into 14 exons that encode the 706 amino acid residues of the protein. The promoter sequence is G + C-rich (67% G + C) with four G/C boxes, it lacks bona fide TATA and CAAT boxes and shows capping site heterogeneity. The existence of pyrimidine-rich motifs, similar to those found in the promoter of ribosomal protein genes, could be relevant to the co-regulation of genes whose products are involved in ribosome biogenesis. Nucleolin contains four RNA binding domains, each about 80 amino acid residues long, which include the 11-residue core ribonucleoprotein consensus motif. Each domain is encoded by two exons, with an intervening sequence interrupting the conserved core motif at roughly the same amino acid position. This latter result suggests that the RNA binding domains are composed of two independent subdomains, whose functions remain to be determined.     

Critical residues for cofactor binding and catalytic activity in the aminoglycoside resistance methyltransferase Sgm

The 16S rRNA methyltransferase Sgm from "Micromonospora zionensis" confers resistance to aminoglycoside antibiotics by specific modification of the 30S ribosomal A site. Sgm is a member of the FmrO family, distant relatives of the S-adenosyl-L-methionine (SAM)-dependent RNA subfamily of methyltransferase enzymes. Using amino acid conservation across the FmrO family, seven putative key amino acids were selected for mutation to assess their role in forming the SAM cofactor binding pocket or in methyl group transfer. Each mutated residue was found to be essential for Sgm function, as no modified protein could effectively support bacterial growth in liquid media containing gentamicin or methylate 30S subunits in vitro. Using isothermal titration calorimetry, Sgm was found to bind SAM with a K(D) (binding constant) of 17.6 microM, and comparable values were obtained for one functional mutant (N179A) and four proteins modified at amino acids predicted to be involved in catalysis in methyl group transfer. In contrast, none of the G135, D156, or D182 Sgm mutants bound the cofactor, confirming their role in creating the SAM binding pocket. These results represent the first functional characterization of any FmrO methyltransferase and may provide a basis for a further structure-function analysis of these aminoglycoside resistance determinants.     

30S ribosomal subunits with fragmented 16S RNA: a new approach for structure and function study of ribosomes.

A new approach for function and structure study of ribosomes based on oligodeoxyribonucleotide-directed cleavage of rRNA with RNase H and subsequent reconstitution of ribosomal subunits from fragmented RNA has been developed. The E coli 16S rRNA was cleaved at 9 regions belonging to different RNA domains. The deletion of 2 large regions was also produced by cleaving 16S rRNA in the presence of 2 or 3 oligonucleotides complementary to different RNA sites. Fragmented and deleted RNA were shown to be efficiently assembled with total ribosomal protein into 30S-like particles. The capacity to form 70S ribosomes and translate both synthetic and natural mRNA of 30S subunits reconstituted from intact and fragmented 16S mRNA was compared. All 30S subunits assembled with fragmented 16S rRNA revealed very different activity: the fragmentation of RNA at the 781-800 and 1392-1408 regions led to the complete inactivation of ribosomes, whereas the RNA fragmentation at the regions 296-305, 913-925, 990-998, 1043-1049, 1207-1215, 1499-1506, 1530-1539 did not significantly influence the ribosome protein synthesis activity, although it was also reduced. These findings are mainly in accordance with the data on the functional activity of some 16S rRNA sites obtained by other methods. The relations between different 16S RNA functional sites are discussed.     

Impact of methylations of m2G966/m5C967 in 16S rRNA on bacterial fitness and translation initiation

The functional centers of the ribosome in all organisms contain ribosomal RNA (rRNA) modifications, which are introduced by specialized enzymes and come at an energy cost for the cell. Surprisingly, none of the modifications tested so far was essential for growth and hence the functional role of modifications is largely unknown. Here, we show that the methyl groups of nucleosides m(2)G966 and m(5)C967 of 16S rRNA in Escherichia coli are important for bacterial fitness. In vitro analysis of all phases of translation suggests that the m(2)G966/m(5)C967 modifications are dispensable for elongation, termination and ribosome recycling. Rather, the modifications modulate the early stages of initiation by stabilizing the binding of fMet-tRNA(fMet) to the 30S pre-initiation complex prior to start-codon recognition. We propose that the m(2)G966 and m(5)C967 modifications help shaping the bacterial proteome, most likely by fine-tuning the rates that determine the fate of a given messenger RNA (mRNA) at early checkpoints of mRNA selection.     

Eukaryotic ribosomal RNA determinants of aminoglycoside resistance and their role in translational fidelity

Recent studies of prokaryotic ribosomes have dramatically increased our knowledge of ribosomal RNA (rRNA) structure, functional centers, and their interactions with antibiotics. However, much less is known about how rRNA function differs between prokaryotic and eukaryotic ribosomes. The core decoding sites are identical in yeast and human 18S rRNAs, suggesting that insights obtained in studies with yeast rRNA mutants can provide information about ribosome function in both species. In this study, we examined the importance of key nucleotides of the 18S rRNA decoding site on ribosome function and aminoglycoside susceptibility in Saccharomyces cerevisiae cells expressing homogeneous populations of mutant ribosomes. We found that residues G577, A1755, and A1756 (corresponding to Escherichia coli residues G530, A1492, and A1493, respectively) are essential for cell viability. We also found that residue G1645 (A1408 in E. coli) and A1754 (G1491 in E. coli) both make significant and distinct contributions to aminoglycoside resistance. Furthermore, we found that mutations at these residues do not alter the basal level of translational accuracy, but influence both paromomycin-induced misreading of sense codons and readthrough of stop codons. This study represents the most comprehensive mutational analysis of the eukaryotic decoding site to date, and suggests that many fundamental features of decoding site function are conserved between prokaryotes and eukaryotes.     

Sequence-structure-function relationships of Tgs1, the yeast snRNA/snoRNA cap hypermethylase

The Saccharomyces cerevisiae Tgs1 methyltransferase (MTase) is responsible for conversion of the m⁷G caps of snRNAs and snoRNAs to a 2,2,7- trimethylguanosine structure. To learn more about the evolutionary origin of Tgs1 and to identify structural features required for its activity, we performed a structure–function study. By using sequence comparison and phylogenetic analysis, we found that Tgs1 shows strongest similarity to Mj0882, a protein related to a family comprised of bacterial rRNA:m²G MTases RsmC and RsmD. The structural information of Mj0882 was used to build a homology model of Tgs1p which allowed us to predict the range of the minimal globular MTase domain and the localization of other residues that may be important for enzyme function. To further characterize functional domains of Tgs1, mutants were constructed and tested for their effects on cell viability, subcellular localization and binding to the small nuclear ribonucleoproteins (snRNPs) and small nucleolar RNPs (snoRNPs). We found that the N-terminal domain of the hypermethylase is dispensable for binding to the common snRNPs and snoRNPs proteins but essential for correct nucleolar localization. Site- directed mutagenesis of Tgs1 allowed also the identification of the residues likely to be involved in the formation of the m7G-binding site and the catalytic center.