YebU is a m5C methyltransferase specific for 16 S rRNA nucleotide 1407

The rRNAs in Escherichia coli contain methylations at 24 nucleotides, which collectively are important for ribosome function. Three of these methylations are m5C modifications located at nucleotides C967 and C1407 in 16S rRNA and at nucleotide C1962 in 23S rRNA. Bacterial rRNA modifications generally require specific enzymes, and only one m5C rRNA methyltransferase, RsmB (formerly Fmu) that methylates nucleotide C967, has previously been identified. BLAST searches of the E.coli genome revealed a single gene, yebU, with sufficient similarity to rsmB to encode a putative m5C RNA methyltransferase. This suggested that the yebU gene product modifies C1407 and/or C1962. Here, we analysed the E.coli rRNAs by matrix assisted laser desorption/ionization mass spectrometry and show that inactivation of the yebU gene leads to loss of methylation at C1407 in 16 S rRNA, but does not interfere with methylation at C1962 in 23 S rRNA. Purified recombinant YebU protein retains its specificity for C1407 in vitro, and methylates 30 S subunits (but not naked 16 S rRNA or 70 S ribosomes) isolated from yebU knockout strains. Nucleotide C1407 is located at a functionally active region of the 30 S subunit interface close to the P site, and YebU-directed methylation of this nucleotide seems to be conserved in bacteria. The yebU knockout strains display slower growth and reduced fitness in competition with wild-type cells. We suggest that a more appropriate designation for yebU would be the rRNA small subunit methyltransferase gene rsmF, and that the nomenclature system be extended to include the rRNA methyltransferases that still await identification.     

Identification and role of functionally important motifs in the 970 loop of Escherichia coli 16S ribosomal RNA

The 970 loop (helix 31) of Escherichia coli 16S ribosomal RNA contains two modified nucleotides, m²G966 and m⁵C967. Positions A964, A969, and C970 are conserved among the Bacteria, Archaea, and Eukarya. The nucleotides present at positions 965, 966, 967, 968, and 971, however, are only conserved and unique within each domain. All organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. Biochemical and structure studies have placed this loop near the P site and have shown it to be involved in the decoding process and in binding the antibiotic tetracycline. To identify the functional components of this ribosomal RNA hairpin, the eight nucleotides of the 970 loop of helix 31 were subjected to saturation mutagenesis and 107 unique functional mutants were isolated and analyzed. Nonrandom nucleotide distributions were observed at each mutated position among the functional isolates. Nucleotide identity at positions 966 and 969 significantly affects ribosome function. Ribosomes with single mutations of m²G966 or m⁵C967 produce more protein in vivo than do wild-type ribosomes. Overexpression of initiation factor 3 specifically restored wild-type levels of protein synthesis to the 966 and 967 mutants, suggesting that modification of these residues is important for initiation factor 3 binding and for the proper initiation of protein synthesis.     

A paradigm for local conformational control of function in the ribosome: binding of ribosomal protein S19 to Escherichia coli 16S rRNA in the presence of S7 is required for methylation of m2G966 and blocks methylation of m5C967 by their respective methyltransferases.

We have partially purified two 16S rRNA-specific methyltransferases, one of which forms m2G966 (m2G MT), while the other one makes m5C967 (m5C MT). The m2G MT uses unmethylated 30S subunits as a substrate, but not free unmethylated 16S rRNA, while the m5C MT functions reciprocally, using free rRNA but not 30S subunits (Nègre, D., Weitzmann, C. and Ofengand, J. (1990) UCLA Symposium: Nucleic Acid Methylation (Alan Liss, New York), pp. 1-17). We have now determined the basis for this unusual inverse specificity at adjacent nucleotides. Binding of ribosomal proteins S7, S9, and S19 to unmodified 16S rRNA individually and in all possible combinations showed that S7 plus S19 were sufficient to block methylation by the m5C MT, while simultaneously inducing methylation by the m2G MT. A purified complex containing stoichiometric amounts of proteins S7, S9, and S19 bound to 16S rRNA was isolated and shown to possess the same methylation properties as 30S subunits, that is, the ability to be methylated by the m2G MT but not by the m5C MT. Since binding of S19 requires prior binding of S7, which had no effect on methylation when bound alone, we attribute the switch in methylase specificity solely to the presence of RNA-bound S19. Single-omission reconstitution of 30S subunits deficient in S19 resulted in particles that could not be efficiently methylated by either enzyme. Thus while binding of S19 is both necessary and sufficient to convert 16S rRNA into a substrate of the m2G MT, binding of either S19 alone or some other protein or combination of proteins to the 16S rRNA can abolish activity of the m5C MT. Binding of S19 to 16S rRNA is known to cause local conformational changes in the 960-975 stem-loop structure surrounding the two methylated nucleotides (Powers, T., Changchien, L.-M., Craven, G. and Noller, H.F. (1988) J. Mol. Biol. 200, 309-319). Our results show that the two ribosomal RNA MTs studied in this work are exquisitely sensitive to this small but nevertheless functionally important structural change.     

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.     

Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding

The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.     

Decreased requirement for 4.5S RNA in 16S and 23S rRNA mutants of Escherichia coli

4.5S RNA is the bacterial homolog of the mammalian signal recognition particle (SRP) RNA that targets ribosome-bound nascent peptides to the endoplasmic reticulum. To explore the interaction of bacterial SRP with the ribosome, we have isolated rRNA suppressor mutations in Escherichia coli that decrease the requirement for 4.5S RNA. Mutations at C732 in 16S rRNA and at A1668 and G1423 in 23S rRNA altered the cellular responses to decreases in both Ffh (the bacterial homolog of SRP54) and 4.5S RNA levels, while the C1066U mutation in 16S rRNA and G424A mutation in 23S rRNA affected the requirement for 4.5S RNA only. These data are consistent with a dual role for 4.5S RNA, one involving co-translational protein secretion by a 4.5S-Ffh complex, the other involving free 4.5S RNA.     

Nucleotides of 18S rRNA surrounding mRNA codons at the human ribosomal A, P, and E sites: a crosslinking study with mRNA analogs carrying an aryl azide group at either the uracil or the guanine residue

The 18S rRNA environment of the mRNA at the decoding site of human 80S ribosomes has been studied by cross-linking with derivatives of hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group either at the N7 atom of the guanine or at the C5 atom of the 5'-terminal uracil residue. The location of the codons on the ribosome at A, P, or E sites has been adjusted by the cognate tRNAs. Three types of complexes have been obtained for each type derivative, namely, (1) codon UUU and Phe-tRNAPhe at the P site (codon GUU at the A site), (2) codon UUU and tRNAPhe at the P site and PheVal-tRNAVal at the A site, and (3) codon GUU and Val-tRNAVal at the P site (codon UUU at the E site). This allowed the placement of modified nucleotides of the mRNA analog at positions -3, +1, or +4 on the ribosome. Mild UV irradiation resulted in tRNA-dependent crosslinking of the mRNA analogs to the 18S rRNA. Nucleotide G961 crosslinked to mRNA position -3, nucleotide G1207 to position +1, and A1823 together with A1824 to position +4. All of these nucleotides are located in the most strongly conserved regions of the small subunit RNA structure, and correspond to nucleotides G693, G926, G1491, and A1492 of bacterial 16S rRNA. Three of them (with the exception of G1491) had been found earlier at the 70S ribosomal decoding site. The similarities and differences between the 16S and 18S rRNA decoding sites are discussed.     

Ribosome-small-subunit-dependent GTPase interacts with tRNA-binding sites on the ribosome

RsgA (ribosome-small-subunit-dependent GTPase A, also known as YjeQ) is a unique GTPase in that guanosine triphosphate hydrolytic activity is activated by the small subunit of the ribosome. Disruption of the gene for RsgA from the genome affects the growth of cells, the subunit association of the ribosome, and the maturation of 16S rRNA. To study the interaction of Escherichia coli RsgA with the ribosome, chemical modifications using dimethylsulfate and kethoxal were performed on the small subunit in the presence or in the absence of RsgA. The chemical reactivities at G530, A790, G925, G926, G966, C1054, G1339, G1405, A1413, and A1493 in 16S rRNA were reduced, while those at A532, A923, G1392, A1408, A1468, and A1483 were enhanced, by the addition of RsgA, together with 5′-guanylylimidodiphosphate. Among them, the chemical reactivities at A532, A790, A923, G925, G926, C1054, G1392, A1413, A1468, A1483, and A1493 were not changed when RsgA was added together with GDP. These results indicate that the binding of RsgA induces conformational changes around the A site, P site, and helix 44, and that guanosine triphosphate hydrolysis induces partial conformational restoration, especially in the head, to dissociate RsgA from the small subunit. RsgA has the capacity to coexist with mRNA in the ribosome while it promotes dissociation of tRNA from the ribosome.     

Role of the Ribosomal P-Site Elements of m2G966, m5C967, and the S9 C-Terminal Tail in Maintenance of the Reading Frame during Translational Elongation in Escherichia coli

The ribosomal P-site hosts the peptidyl-tRNAs during translation elongation. Which P-site elements support these tRNA species to maintain codon-anticodon interactions has remained unclear. We investigated the effects of P-site features of methylations of G966, C967, and the conserved C-terminal tail sequence of Ser, Lys, and Arg (SKR) of the S9 ribosomal protein in maintenance of the translational reading frame of an mRNA. We generated Escherichia coli strains deleted for the SKR sequence in S9 ribosomal protein, RsmB (which methylates C967), and RsmD (which methylates G966) and used them to translate LacZ from its +1 and −1 out-of-frame constructs. We show that the S9 SKR tail prevents both the +1 and −1 frameshifts and plays a general role in holding the P-site tRNA/peptidyl-tRNA in place. In contrast, the G966 and C967 methylations did not make a direct contribution to the maintenance of the translational frame of an mRNA. However, deletion of rsmB in the S9Δ3 background caused significantly increased −1 frameshifting at 37°C. Interestingly, the effects of the deficiency of C967 methylation were annulled when the E. coli strain was grown at 30°C, supporting its context-dependent role.     

A cluster of methylations in the domain IV of 25S rRNA is required for ribosome stability

In all three domains of life ribosomal RNAs are extensively modified at functionally important sites of the ribosome. These modifications are believed to fine-tune the ribosome structure for optimal translation. However, the precise mechanistic effect of modifications on ribosome function remains largely unknown. Here we show that a cluster of methylated nucleotides in domain IV of 25S rRNA is critical for integrity of the large ribosomal subunit. We identified the elusive cytosine-5 methyltransferase for C2278 in yeast as Rcm1 and found that a combined loss of cytosine-5 methylation at C2278 and ribose methylation at G2288 caused dramatic ribosome instability, resulting in loss of 60S ribosomal subunits. Structural and biochemical analyses revealed that this instability was caused by changes in the structure of 25S rRNA and a consequent loss of multiple ribosomal proteins from the large ribosomal subunit. Our data demonstrate that individual RNA modifications can strongly affect structure of large ribonucleoprotein complexes.     

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.     

Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli

Many contacts between the ribosome and its principal substrates, tRNA and mRNA, involve universally conserved rRNA nucleotides, implying their functional importance in translation. Here, we measure the in vivo translation activity conferred by substitution of each 16S rRNA base believed to contribute to the A or P site. We find that the 30S P site is generally more tolerant of mutation than the 30S A site. In the A site, A1493C or any substitution of G530 or A1492 results in complete loss of translation activity, while A1493U and A1493G decrease translation activity by >20-fold. Among the P-site nucleotides, A1339 is most critical; any mutation of A1339 confers a >18-fold decrease in translation activity. Regarding all other P-site bases, ribosomes harboring at least one substitution retain considerable activity, >10% that of control ribosomes. Moreover, several sets of multiple substitutions within the 30S P site fail to inactivate the ribosome. The robust nature of the 30S P site indicates that its interaction with the codon-anticodon helix is less stringent than that of the 30S A site. In addition, we show that G1338A suppresses phenotypes conferred by m(2)G966A and several multiple P-site substitutions, suggesting that adenine at position 1338 can stabilize tRNA interaction in the P site.     

Yeast Rrp8p,a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA

Ribosomal RNA undergoes various modifications to optimize ribosomal structure and expand the topological potential of RNA. The most common nucleotide modifications in ribosomal RNA (rRNA) are pseudouridylations and 2′-O methylations (Nm), performed by H/ACA box snoRNAs and C/D box snoRNAs, respectively. Furthermore, rRNAs of both ribosomal subunits also contain various base modifications, which are catalysed by specific enzymes. These modifications cluster in highly conserved areas of the ribosome. Although most enzymes catalysing 18S rRNA base modifications have been identified, little is known about the 25S rRNA base modifications. The m¹A modification at position 645 in Helix 25.1 is highly conserved in eukaryotes. Helix formation in this region of the 25S rRNA might be a prerequisite for a correct topological framework for 5.8S rRNA to interact with 25S rRNA. Surprisingly, we have identified ribosomal RNA processing protein 8 (Rrp8), a nucleolar Rossman-fold like methyltransferase, to carry out the m¹A base modification at position 645, although Rrp8 was previously shown to be involved in A2 cleavage and 40S biogenesis. In addition, we were able to identify specific point mutations in Rrp8, which show that a reduced S-adenosyl-methionine binding influences the quality of the 60S subunit. This highlights the dual functionality of Rrp8 in the biogenesis of both subunits.     

Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16 S rRNA

Transfer RNA protects a characteristic set of bases in 16 S rRNA from chemical probes when it binds to ribosomes. We used several criteria, based on construction of well-characterized in vitro ribosome-tRNA complexes, to assign these proteins to A or P-site binding. All of these approaches lead to similar conclusions. In the A site, tRNA caused protection of G529, G530, A1492 and A1493 (strongly), and A1408 and G1494 (weakly). In the P site, the protected bases are G693, A794, C795, G926 and G1401 (strong), and A532, G966, G1338 and G1339 (weak). In contrast to what is observed for 23 S rRNA, blocking the release of EF-Tu.GDP from the ribosome by kirromycin has no detectable effect on the protection of bases in 16 S rRNA.     

Cysteine of sequence motif VI is essential for nucleophilic catalysis by yeast tRNA m5C methyltransferase

Sequence comparison of several RNA m(5)C methyltransferases identifies two conserved cysteine residues that belong to signature motifs IV and VI of RNA and DNA methyltransferases. While the cysteine of motif IV is used as the nucleophilic catalyst by DNA m(5)C methyltransferases, this role is fulfilled by the cysteine of motif VI in Escherichia coli 16S rRNA m(5)C967 methyltransferase, but whether this conclusion applies to other RNA m(5)C methyltransferases remains to be verified. Yeast tRNA m(5)C methyltransferase Trm4p is a multisite-specific S-adenosyl-L-methionine-dependent enzyme that catalyzes the methylation of cytosine at C5 in several positions of tRNA. Here, we confirm that Cys310 of motif VI in Trm4p is essential for nucleophilic catalysis, presumably by forming a covalent link with carbon 6 of cytosine. Indeed, the enzyme is able to form a stable covalent adduct with the 5-fluorocytosine-containing RNA substrate analog, whereas the C310A mutant protein is inactive and unable to form the covalent complex.     

Chemical synthesis of capped RNA fragments and their ability to complex with eukaryotic ribosomes

In other to examine the binding ability of the 5'-terminal part of eukaryotic mRNA to 80S ribosome, several kinds of oligoribonucleotides, pA-U-G, m7G5'pppA-U-G, m7G5'pppG-U, m7G5'pppA-U-G-A-C-C, were synthesized chemically. The binding experiments of oligonucleotides to 80S ribosome showed that the capped structure as well as AUG are essential for ribosome binding, and the efficiency is enhanced by the 5'-leader sequence if it would include complementary sequence to the 3'-terminal part of 18S rRNA.     

Exposition of a family of RNA m(5)C methyltransferases from searching genomic and proteomic sequences.

The Escherichia coli fmu gene product has recently been determined to be the 16S rRNA m(5)C 967 methyltransferase. As such, Fmu represents the first protein identified as an S -adenosyl-L-methionine (AdoMet)- dependent RNA m(5)C methyltransferase whose amino acid sequence is known. Using the amino acid sequence of Fmu as an initial probe in an iterative search of completed DNA sequence databases, 27 homologous ORF products were identified as probable RNA m(5)C methyltransferases. Further analysis of sequences in undeposited genomic sequencing data and EST databases yielded more than 30 additional homologs. These putative RNA m(5)C methyltransferases are grouped into eight subfamilies, some of which are predicted to consist of direct genetic counterparts, or orthologs. The enzymes proposed to be RNA m(5)C methyltransferases have sequence motifs closely related to signature sequences found in the well-studied DNA m(5)C methyltransferases and other AdoMet-dependent methyltransferases. Structure-function correlates in the known AdoMet methyltransferases support the assignment of this family as RNA m(5)C methyltransferases.