We have developed a rapid PCR-oligonucleotide ligation assay that can discriminate single base substitutions that are associated with clarithromycin resistance in Helicobacter pylori. Susceptible isolates were wild type at positions 2143 and 2144 (cognate to 2058 and 2059 in Escherichia coli), while 93% of the resistant isolates contained A-to-G mutations at either position and 7% of the isolates contained A-to-C mutations at position 2143. In addition, the MIC for 86% of the resistant isolates with an A2143 mutation was , or = 64 micrograms per ml, and that for 89% of the resistant isolates with an A2144 mutation was , or = 32 micrograms per ml. ...
Genetic information processingProtein synthesistRNA and rRNA base modification16S rRNA (guanine(966)-N(2))-methyltransferase RsmD (TIGR00095; EC 2.1.1.171; HMM-score: 12.5) ...
Genetic information processingProtein synthesistRNA and rRNA base modification16S rRNA (cytosine(967)-C(5))-methyltransferase (TIGR00563; EC 2.1.1.176; HMM-score: 27.7) ...
Chen, H.,Lim, C.K.,Lee, Y.K.,Chan, Y.N. (2000). Comparative analysis of the genes encoding 23S-5S rRNA intergenic spacer regions of Lactobacillus casei-related strains. International Journal of Systematic and Evolutionary Microbiology 50 (2) : 471-478. [email protected] Repository ...
Staphylococcus aureus is an important human and animal pathogen, and is regarded as an important cause of intramammary infection (IMI) in ruminants. Staphylococcus aureus genetic variability and virulence factors have been well studied in veterinary medicine, especially in cows as support for control and management of IMI. The aim of the present study was to genotype 71 Staph. aureus isolates from the bulk tank and foremilk of water buffaloes (n. = 40) and from udder tissue (n. = 7) and foremilk (n. = 24) from small ruminants. The method used was previously applied to bovine Staph. aureus and is based on the amplification of the 16S-23S rRNA intergenic spacer region. The technique applied was able to identify different Staph. aureus genotypes isolated from dairy species other than the bovine species, and cluster the genotypes according to species and herds. Virulence gene distribution was consistent with genotype differentiation. The isolates were also characterized through determination of the ...
NCTC <- H.R. Carne, 84. Pig. Type strain. Taxonomy/description (1300, 2837, 2838, 2839, 6728, 16013). Sequence accession no. 16S rRNA gene: X79225, 16S rRNA gene, 16S-23S rRNA intergenic spacer: EU194563. Murein: A11.53. (Medium 104, 37°C, anaerobic ...
Onsensus sequence, missing both most important nucleotides G, after the methionine codon and A, three nucleotides before the methionine that determine the
Linezolid resistance in the absence of oxazolidinone exposure and surveillance.As described previously, MRSA CM-05 did not exhibit any mutations in domain V of the 23S rRNA (28). The identification of the cfr gene, which encodes an rRNA methylase, in this isolate recovered from a patient after such a short exposure to linezolid (28) indicates that the gene was also most likely acquired by the isolate under a selective pressure that did not involve exposure to oxazolidinones. An alternative explanation is that the strain was selected in an unidentified patient exposed to linezolid and was then passed on to the case patient. However, we did not find any evidence of the presence of cfr in any of the clinical isolates of MRSA previously recovered in Colombia or isolated around the time that the first isolate was discovered (see below), making this alternative possibility less likely. Although linezolid resistance in the absence of oxazolidinone exposure had been documented in Enterococcus spp. (2, ...
MRM1, 50 µg. Mitochondrial rRNA methyltransferase 1 homolog, also known as MRM1, probably methylates the ribose of guanosine G-2270 in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S).
The 2,030 isolates analyzed in the present study comprised 1,235 isolates from stool samples from hospital patients, 395 isolates from stool samples referred via community practitioners, 150 isolates from the hospital environment, 27 isolates from veterinary sources, 89 isolates from the general environment, 1 isolate from an extraintestinal human site, and 133 reference strains held in the National Collection of Type Cultures, the American Type Culture Collection and the Culture Collection, University of Göteborg, and in the personal collections of C. difficile types held by Delmee and others (6, 16) and other members of the International Study Group on C. difficile (3).. PCR ribotyping was performed in duplicate, with slight modifications to a method described previously (13). Briefly, bacteria were harvested from overnight anaerobic cultures on Fastidious Anaerobe Agar (LabM, Bury, United Kingdom) supplemented with 6% horse blood. Crude template nucleic acid was prepared by resuspension of ...
In Korea, Mycoplasma pneumoniae was detected in 255/2,089 respiratory specimens collected during 2000-2011; 80 isolates carried 23S rRNA gene mutations, and 69/123 culture-positive samples with the mutation were resistant to 5 macrolides. During 2000-2011, prevalence of the mutation increased substantially. These findings have critical implications for the treatment of children with mycoplasma pneumonia.
In order to assess the frequency of clinically relevant linezolid-resistant staphylococcal isolates, and the role of linezolid in maintaining and coselecting multiple resistance mechanisms (cfr, 23S rRNA, L3/L4 mutations), a prospective Italian study was performed from 2010 to 2011 to confirm the diffusion of three major multidrug-resistant clones (ST2, ST5, ST23).
The peptidyl transferase center (PTC) is located in a protein free environment, thus confirming that the ribosome is a ribozyme. This arched void has dimensions suitable for accommodating the 3ends of the A-and the P-site tRNAs, and is situated within a universal sizable symmetry-related region that connects all ribosomal functional centers involved in amino-acid polymerization. The linkage between the elaborate PTC architecture and the A-site tRNA position revealed that the A-to P-site passage of the tRNA 3end is performed by a rotatory motion, which leads to stereochemistry suitable for peptide bond formation and for substrate mediated catalysis, thus suggesting that the PTC evolved by genefusion. Adjacent to the PTC is the entrance of the protein exit tunnel, shown to play active roles in sequence-specific gating of nascent chains and in responding to cellular signals. This tunnel also provides a site that may be exploited for local co-translational folding and seems to assist in nascent ...
Under the intluence of 5-tluoro-uridine, the ultrastructure of the rDNA transcription units in Xenopus oocytes is altered. Whereas part of the matrix units maintains anormal aspect or shows various degrees of inhibition, in a strong proportion of the transcription units the alternating pattern of matrix units and fibril-free spacer regions is no longer recognized. Transcriptional complexes are found along the entire DNP axis, including the regions of the spacers. These observations support biochemical data on transcription in rDNA spacer region ...
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We evaluated isolates obtained from children with Mycoplasma pneumoniae infection throughout Japan during 2008-2015. The highest prevalence of macrolide-resistant M. pneumoniae was 81.6% in 2012, followed by 59.3% in 2014 and 43.6% in 2015. The prevalence of macrolide-resistant M. pneumoniae among children in Japan has decreased.
Interpretive Summary: Technical Abstract: Primers were designed that selectively amplified by the polymerase chain reaction (PCR) a region of the 16S-23S rDNA intergenic spacer region (ISR) unique to Flavobacterium columnare. The primers were sensitive, capable of detecting 7 colony forming units, and specific for F. columnare. A second goal was to examine horizontal transmission of F. columnare in channel catfish. Two experiments were conducted. In the first, channel catfish fingerlings were injected intramuscularly with F. columnare. Positive detection of F. columnare in tank water occurred 3 d post-challenge for both PCR and traditional culture techniques; however, a greater proportion of tanks were positive for F. columnare by PCR detection. In a second experiment, fingerlings were challenged by immersion bath. F. columnare was detected in water by PCR and culture techniques 1 and 2 d post-challenge, respectively. In both experiments, PCR detection in tissues was superior to traditional ...
Ribosomal DNA (rDNA) of plants is present in high copy number and shows variation between and within species in the length of the intergenic spacer (IGS). The 45S rDNA of flowering plants includes the 5.8S, 18S and 25S rDNA genes, the internal transcribed spacer (ITS1 and ITS2), and the intergenic spacer 45S-IGS (25S-18S). This study identified six different types of 45S-IGS, A to F, which at 363 bp, 1121 bp, 1717 bp, 1969 bp, 2036 bp and 2111 bp in length, respectively, were much shorter than the reported reference IGS sequences in B. oleracea var. alboglabra. The shortest two IGS types, A and B, lacked the transcription initiation site, non-transcribed spacer, and external transcribed spacer. Functional behavior of those two IGS types in relation to rRNA synthesis is a subject of further investigation. The other four IGSs had subtle variations in the transcription termination site, guanine-cytosine (GC) content, and number of tandem repeats, but the external transcribed spacers of these four IGSs were
A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100-1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species
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Translational GTPases (trGTPases) are proteins in which the GTPase activity is induced by the large ribosomal subunit [1, 2]. Several members of this protein family (EF-G, EF-Tu, IF2 and RF3) bind to an overlapping site on the ribosome [1, 3-6]. This conserved region of the large subunit includes part of domain II of 23S RNA (the binding site for the antibiotic thiostreptone), part of domain VI (the sarcin-ricin loop), and proteins L11 and L7/12. This region is responsible for activating the trGTPases [1, 2].. The specific sequence features of the trGTPases allow proteins that belong to this family to be identified [7]. In bacteria, the family includes proteins that are considered to belong to the "classical" set of translational GTPases (EF-G, EF-Tu, IF2, RF3), proteins that bind to the ribosome and have auxiliary or unidentified functions (SelB, Tet, LepA, TypA), and a group of proteins that have acquired functions in sulfur metabolism and might have lost their ability to bind to the ribosome ...
F.SCHLUNZEN,R.ZARIVACH,J.HARMS,A.BASHAN,A.TOCILJ, R.ALBRECHT,A.YONATH,F.FRANCESCHI. STRUCTURAL BASIS FOR THE INTERACTION OF ANTIBIOTICS WITH THE PEPTIDYL TRANSFERASE CENTRE IN EUBACTERIA.. NATURE V. 413 814 2001 ASTM NATUAS UK ISSN 0028-0836 ...
The GCC region is a small but important part of the global economy. Its 2007 real GDP was around US$810 billion, accounting for just 2.1% of the global total 12 . Despite the relatively small size of their economies and populations13 , the rate of GDP growth in GCC countries reached 6.4% in 2007, which is similar to the Asia Pacific average (5% to 6%). Its growth rate exceeded the averages for Latin America, Europe, the OECD and the Middle East. In terms of GDP per capita, Qatar, the UAE, and Kuwait rank among the highest... ...
Thiostrepton is a natural cyclic oligopeptide antibiotic of the thiopeptide class, derived from several strains of streptomycetes, such as Streptomyces azureus and Streptomyces laurentii. Thiostrepton is a natural product of the ribosomally synthesized and post-translationally modified peptide (RiPP) class. Thiostrepton was discovered by Donovick et al. who described its antibacterial properties in 1955. Dorothy Crowfoot Hodgkin solved the structure of thiostrepton in 1970. Early in 1978, Bycroft and Gowland proposed the biosynthesis of thiostrepton, which was still unclear until 2009. Several studies of thiopeptide biosynthesis have been contemporarily published in 2009 and two of them (Liao et al. and Kelly et al.) included the similar biosynthesis of thiostrepton: its ribosomally synthesized from thiostrepton biosynthetic genes (tsr genes) and posttranslational modification is needed. A total synthesis of thiostrepton was completed by K.C. Nicolaou, et al. in 2004. Thiostrepton has been used ...
We investigated 16S rRNA methyltransferases in 38 blaNDM-1-positive Pseudomonas aeruginosa isolates and found RmtC in 3 isolates, 1 of which also harbored RmtF. The isolates were clonally unrelated; rmtC and rmtF genes were located on a chromosome with the blaNDM-1 gene. Strategies are needed to limit the spread of such isolates ...
Lactobacillus acetotolerans strain ATCC 43578 16S ribosomal RNA gene,partial sequence; 16S-23S intergenic spacer, complete sequence; and 23Sribosomal RNA gene, partial ...
Rychlik, I., Cerná, J., Chládek, S., Zemlicka, J. and Haladová, Z. (1969). "Substrate specificity of ribosomal peptidyl transferase: 2′(3′)-O-aminoacyl nucleosides as acceptors of the peptide chain on the amino acid site". J. Mol. Biol. 43: 13-24. PMID 4897787. ...
Lactobacillus fructivorans strain ATCC 8288 16S ribosomal RNA gene, partialsequence; 16S-23S intergenic spacer, complete sequence; and 23S ribosomalRNA gene, partial ...
Synthetic biology technology could lead to new antibiotics, modified protein-generators. Synthetic biology researchers at Northwestern University, working with partners at Harvard Medical School, have for the first time synthesized ribosomes -- cell structures responsible for generating all proteins and enzymes in our bodies -- from scratch in a test tube.. Others have previously tried to synthesize ribosomes from their constituent parts, but the efforts have yielded poorly functional ribosomes under conditions that do not replicate the environment of a living cell. In addition, attempts to combine ribosome synthesis and assembly in a single process have failed for decades.. Michael C. Jewett, a synthetic biologist at Northwestern, George M. Church, a geneticist at Harvard Medical School, and colleagues recently took another approach: they mimicked the natural synthesis of a ribosome, allowing natural enzymes of a cell to help facilitate the man-made construction.. The technology could lead to ...
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The large subunit of the ribosome contains the site at which peptide bonds are formed in the process of translation. Another striking feature of the large subunit is the exit tunnel. This feature begins at the site of peptide bond formation traversing 100 angstroms before opening to the cytosolic environment on the opposite side of the large subunit. It has been known for some time that the ribosome exit tunnel is the site of action for MLS antibiotics, one such example being erythromycin. More recently the exit tunnel has been shown to be involved in sensing and regulating the egress of newly synthesized peptides. As the exact mechanisms by which either macrolides such as erythromycin or nascent peptides inhibit ribosome function is not known, understanding how both of these regulatory activities are accomplished remains an important challenge in understanding ribosome structure and function. Through mutational analysis and the use of translational reporters, I have obtained results which show ...
This, my Cand. Scient. (MS) project, was completed at the Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen. My supervisor was Associate Professor Birte Vester who was part of the RNA group led by Professor Roger Garrett. Here I did three years of lab-work investigating posttranscriptional modifications of ribosomal RNA using standard lab methods combined with mass spectrometry. I was able to isolate the part of the 23S ribosomal RNA that constitutes the peptidyl transferase centre using site-directed RNaseH digestion followed by isolation with PAGE. The isolated fragments were analysed by MALDI-MS, where I was so fortunate to collaborate with Associate Professor Finn Kirpekar (University of Southern Denmark) who analyzed the fragments - and taught me to perform this type of analyzis on equipment in Copenhagen. I also screened these fragments for the mass-silent pseudouridines using chemical modification and detection with RT-PCR and visualization on ...
RS18_THETH] Binds as a heterodimer with protein S6 to the central domain of the 16S rRNA, where it helps stabilize the platform of the 30S subunit (By similarity). [RS4_THET8] One of the primary rRNA binding proteins, it binds directly to 16S rRNA where it helps nucleate assembly of the body and platform of the 30S subunit. Binds mRNA in the 70S ribosome, positioning it for translation.[HAMAP-Rule:MF_01306_B] [RS14Z_THETH] Binds 16S rRNA, required for the assembly of 30S particles and may also be responsible for determining the conformation of the 16S rRNA at the A site (By similarity). [RS15_THETH] One of the primary rRNA binding proteins, it binds directly to 16S rRNA where it helps nucleate assembly of the platform of the 30S subunit by binding and bridging several RNA helices of the 16S rRNA. Forms an intersubunit bridge (bridge B4) with the 23S rRNA of the 50S subunit in the ribosome (By similarity). [RS13_THET8] Located at the top of the head of the 30S subunit, it contacts several helices ...
The Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) in England and Wales has monitored azithromycin resistance since 2001. In 2007, high-level azithromycin resistance (MICs >256 mg/L) was identified for the first time in six isolates, all of which were the same sequence type (ST 649).
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Summary Different PCR-based DNA fingerprinting techniques were evaluated for typing 26 clinical isolates belonging to the Acinetobacter calcoaceticus-A. baumannii complex. Seven isolates belonged to a previously defined outbreak while 19 isolates were unrelated epidemiologically. The PCR-based DNA fingerprinting techniques used were: (i) repetitive extragenic palindromic (REP) PCR; (ii) enterobacterial repetitive intergenic consensus (ERIC) PCR; (iii) randomly amplified polymorphic DNA with M13 forward primer; (iv) restriction analysis of the amplified 16S rRNA gene (ARDRA-16S); and (v) restriction analysis of an amplified region containing the 16S-23S rRNA spacer region and part of the 23S rRNA gene (ARDRA 23S + spacer). The discrimination index for the PCR-based DNA fingerprinting techniques was: 0.99 for REP; 0.94 for ERIC; 0.87 for M13; 0.60 for ARDRA-16S digested with Hpa II and |0.50 for ARDRA 23S + spacer. It was concluded that REP-PCR possessed high discriminatory power and reproducibility in
Huanglongbing (HLB) is a destructive disease of citrus production worldwide. All known commercial citrus cultivars are susceptible to HLB. The disease was first noted in Chaoshan area in Guangdong Province of the Peoples Republic of China in the late of 1800s [1] and is currently distributed in 10 citrus producing provinces in South China. HLB is now established in Sao Paulo of Brazil [2] and Florida of the United States [3] where it poses a great threat to the citrus industry. The disease is associated with three species of non-culturable, phloem-limited, α-Proteobacteria: Candidatus Liberibacter asiaticus, Ca. L. africanus, and Ca. L. americanus [4, 5]. In both China and U.S., only Ca. L. asiaticus has been detected. Due to the lack of pure culture, Ca. L. asiaticus has been poorly characterized. Little is known about the bacterial biology, genetic diversity, and epidemiology.. Sequence analyses of conserve genomic loci such as 16S rRNA gene and 16S/23S intergenic spacer regions ...
In yeast, guide snoRNAs have been assigned to 51 of the 55 rRNA ribose methylation sites. LSU-Um2918 is one of the four remaining positions. This residue is highly conserved and located in the peptidyl transferase center of the ribosome. The equivalent position on the E. coli 23S rRNA is methylated by FtsJ/RrmJ which has three yeast homologs: Spb1, involved in biogenesis of LSU; Trm7, a tRNA methyltransferase; and Mrm2, a mitochondrial 21S rRNA methyltransferase. We demonstrate that a point mutation in the Ado-Met binding site of Spb1p affects cell growth but does not abolish methylation of U2918. When this mutation is combined with disruption of snR52 (a snoRNA C/D), cell growth is severely impaired and U2918 is no longer methylated. In vitro, Spb1p is able to methylate U2918 on 60S subunits. Our results reveal the importance of this methylation for which two mechanisms coexist: a site-specific methyltransferase (Spb1p) and a snoRNA-dependent mechanism ...
The chloramphenicol (Cm)-inducible cmlA gene of Tn1696 specifies nonenzymatic resistance to Cm and is regulated by attenuation. The first eight codons of the leader specify a peptide that inhibits peptidyl transferase in vitro. Functionally similar, but less inhibitory, peptides are encoded by the leaders of Cm-inducible cat genes. However, the cat and cmlA coding sequences are unrelated and specify proteins of unrelated function. The inhibition of peptidyl transferase by the leader peptides is additive with that of Cm. Erythromycin competes with the inhibitory action of the peptides, and erythromycin and the peptides footprint to overlapping sites at the peptidyl transferase center of 23S rRNA. It is proposed that translation of the cmlA and cat leaders transiently pauses upon synthesis of the inhibitor peptides. The predicted site of pausing is identical to the leader site where long-term occupancy by a ribosome (ribosome stalling) will activate downstream gene expression. We therefore propose ...
canSAR Domains and Structures of Q0TCF6 | rplF | 50S ribosomal protein L6 - Also known as RL6_ECOL5, rplF. This protein binds to the 23S rRNA, and is important in its secondary structure. It is located near the subunit interface in the base of the L7/L12 stalk, and near the tRNA binding site of the peptidyltransferase center. Part of the 50S ribosomal subunit.
Three papers in the 11 Aug. 2000 issue characterized the atomic structure of the large ribosomal subunit, and used that structural underpinning to explore the mechanisms of peptide bond formation in the ribosome. In a comment, Barta et al. question whether the highly conserved nucleotide A2451 is the sole catalytic site, as suggested in the 11 Aug. papers, and argue that metal ion catalysis may remain a viable alternative to the acid-base catalysis model favored in those studies. Berg and Lorsch, in a separate comment, put forth an alternative mechanism for peptide bond formation that they maintain avoids "three significant difficulties" implicit in the mechanism proposed in the 11 Aug. papers. In their response, Nissen et al. critically review the literature cited by Barta et al. and conclude that it does not substantially undermine their proposals; they also present additional information bearing on the three deficiencies alleged by Berg and Lorsch. The full text of these comments can be seen ...
Ribosomes are comprised of 65% RNA and 35% proteins. Ribosomes are cellular organelles that are responsible for Protein Synthesis. Ribosomes function
In the field of synthetic biology the strength of an RBS is defined as the rate at which translation is initiated by the RBS sequence on any given mRNA molecule. Going back a step, we see that translation initiation occurs preferentially at certain mRNA sequences, which show a similarity to the consensus -Shine-Delgarno- sequence. This is due to the optimal binding of the 16S rRNA at these regions. In other words, the RBS strength may loosely be defined as the rate of ribosome binding to any given mRNA molecule. (Click here for more information. For our project it is not directly the binding equilibria of ribosome to mRNA that is of interest to us, but rather the netto rate of protein production. Thus, in our experimentation we define the RBS strength as the production rate of a given protein downstream of an RBS. ...
Valnemulin hydrochloride is a pleuromutilin antibiotic which inhibits protein synthesis in bacteria by binding the peptidyl transferase enzyme in the 50s ribosomal subunit. - Mechanism of Action & Protocol.
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One of her highlights this year was modeling at the United nations during the visit of Princess Nauf Benda Al Saud. She was recently scouted by Ishika Chaudhary to walk in NYFW, LAFW and Paris Fashion week this coming season and will be an ambassador in the upcoming Fashion for Education in Africa which she is very proud of. ...
Antibiotics targeting the bacterial ribosome typically bind to highly conserved rRNA regions with only minor phylogenetic sequence variations. It is unclear whether these sequence variations affect antibiotic susceptibility or resistance development. To address this question, we have investigated the drug binding pockets of aminoglycosides and macrolides/ketolides. The binding site of aminoglycosides is located within helix 44 of the 16S rRNA (A site); macrolides/ketolides bind to domain V of the 23S rRNA (peptidyltransferase center). We have used mutagenesis of rRNA sequences in Mycobacterium smegmatis ribosomes to reconstruct the different bacterial drug binding sites and to study the effects of rRNA sequence variations on drug activity. Our results provide a rationale for differences in species-specific drug susceptibility patterns and species-specific resistance phenotypes associated with mutational alterations in the drug binding pocket. ...
Escherichia coli ribosomal RNA (rRNA) is post-transcriptionally modified by site-specific enzymes. The role of most modifications is not known and little is known about how these enzymes recognize their target substrates. In this thesis, we have structurally and functionally characterized two S-adenosyl-methionine (SAM) dependent 23S rRNA methyltransferases (MTases) that act during the early stages of ribosome assembly in E. coli.. RlmM methylates the 2O-ribose of C2498 in 23S rRNA. We have solved crystal structures of apo RlmM at 1.9Å resolution and of an RlmM-SAM complex at 2.6Å resolution. The RlmM structure revealed an N-terminal THUMP domain and a C-terminal catalytic Rossmann-fold MTase domain. A continuous patch of conserved positive charge on the RlmM surface is likely used for RNA substrate recognition. The SAM-binding site is open and shallow, suggesting that the RNA substrate may be required for tight cofactor binding. Further, we have shown RlmM MTase activity on in vitro ...
Haloarcula marismortui ATCC ® 43049D-5™ Designation: Genomic DNA from Haloarcula marismortui strain DSM 3752 TypeStrain=True Application: