RESUMEN
There are 10 known putative pseudouridine synthase genes in Escherichia coli. The products of six have been previously assigned, one to formation of the single pseudouridine in 16S RNA, three to the formation of seven pseudouridines in 23S RNA, and three to the formation of three pseudouridines in tRNA (one synthase makes pseudouridine in 23S RNA and tRNA). Here we show that the remaining four putative synthase genes make bona fide pseudouridine synthases and identify which pseudouridines they make. RluB (formerly YciL) and RluE (formerly YmfC) make pseudouridine2605 and pseudouridine2457, respectively, in 23S RNA. RluF (formerly YjbC) makes the newly discovered pseudouridine2604 in 23S RNA, and TruC (formerly YqcB) makes pseudouridine65 in tRNA(Ile1) and tRNA(Asp). Deletion of each of these synthase genes individually had no effect on exponential growth in rich media at 25 degrees C, 37 degrees C, or 42 degrees C. A strain lacking RluB and RluF also showed no growth defect under these conditions. Mutation of a conserved aspartate in a common sequence motif, previously shown to be essential for the other six E. coli pseudouridine synthases and several yeast pseudouridine synthases, also caused a loss of in vivo activity in all four of the synthases studied in this work.
Asunto(s)
Escherichia coli/enzimología , Hidroliasas/genética , Seudouridina/biosíntesis , Secuencia de Bases , Sitios de Unión , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Hidroliasas/química , Hidroliasas/fisiología , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN/biosíntesis , ARN Bacteriano/biosíntesisRESUMEN
This laboratory previously showed that truncation of the gene for RluD, the Escherichia coli pseudouridine synthase responsible for synthesis of 23S rRNA pseudouridines 1911, 1915, and 1917, blocks pseudouridine formation and inhibits growth. We now show that RluD mutants at the essential aspartate 139 allow these two functions of RluD to be separated. In vitro, RluD with aspartate 139 replaced by threonine or asparagine is completely inactive. In vivo, the growth defect could be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD with aspartate 139 replaced by threonine or asparagine. Pseudouridine sequencing of the 23S rRNA from these transformed strains demonstrated the lack of these pseudouridines. Pseudoreversion, which has previously been shown to restore growth without pseudouridine formation by mutation at a distant position on the chromosome, was not responsible because transformation with empty vector under identical conditions did not alter the growth rate.
Asunto(s)
Escherichia coli/genética , Hidroliasas/metabolismo , Mutación Puntual , Seudouridina/biosíntesis , ARN Ribosómico 23S/genética , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Escherichia coli/crecimiento & desarrollo , Hidroliasas/genéticaRESUMEN
Pseudouridine is present in ribosomal RNA, transfer RNA, tmRNA, and small nuclear and nucleolar RNAs. All are structured molecules. Pseudouridine is made by enzyme-catalyzed isomerization of specifically selected U residues after the polynucleotide chain is made. No energy input is required. Pseudouridine formation creates a hydrogen bond donor at the equivalent of uridine C-5. Therefore, a major role of pseudouridine may be to strengthen particular RNA conformations and/or RNA-RNA interactions because of this extra H-bond capability. Understanding the role of pseudouridine critically depends on knowledge of their location and number in RNA. The mapping method described here has greatly simplified this task and made it possible to survey many organisms. Procedures are described for mapping pseudouridines in large RNAs like ribosomal RNA and in small RNAs like tRNA. The method involves carbodiimide adduct formation with U, G, and pseudouridine followed by mild alkali to remove the adduct from U and G but not from the N-3 of pseudouridine. This results in attenuation of primed reverse transcription resulting in a stop band one residue 3' to the pseudouridine on sequencing gels. Use of primers means that purified RNAs are not needed, only knowledge of its primary sequence, but limit the sequence scanned to 30-40 residues from the 3' end. A poly(A) tailing procedure is described that allows extension of the method to within a few nucleotides of the 3' terminus.
Asunto(s)
Técnicas Genéticas , Seudouridina/química , Seudouridina/genética , ARN/química , Secuencia de Bases , Cartilla de ADN/química , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Modelos Químicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Poli A , Unión Proteica , ARN/ultraestructura , ARN Ribosómico/química , ARN Nuclear Pequeño/químicaRESUMEN
psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes Um2552 in E. coli, makes the 50S subunit less stable at 1 mM Mg++ (Bügl et al. 2000) and inhibits subunit joining (Caldas et al. 2000), but, in this case, it is not yet known whether the effects are due to the lack of 2'-O-methylation or to the absence of the enzyme itself. Is there any role for the psi residues themselves? First, as noted above, the 3 psi made by RluD which cluster in the end-loop of helix 69 are highly conserved, with one being universal (Fig. 2B). In the 70S-tRNA structure (Yusupov et al. 2001), the loop of this helix containing the psi supports the anticodon arm of A-site tRNA near its juncture with the amino acid arm. The middle of helix 69 does the same thing for P-site tRNA. Unfortunately, the resolution is not yet sufficient to provide a more precise alignment of the psi residues with the other structural elements of the tRNA-ribosome complex so that one cannot yet determine what role, if any, is played by the N-1 H that distinguishes psi from U. Second, and more generally, some psi residues in the LSU appear to be near the site of peptide-bond formation or tRNA binding but not actually at it (Fig. 2B) (Nissen et al. 2000; Yusupov et al. 2001). For example, position 2492 is commonly psi and is only six residues away from A2486, the A postulated to catalyze peptide-bond formation. Position 2589 is psi in all the eukaryotes and is next to 2588, which base-pairs with the C75 of A-site tRNA. Residue 2620, which interacts with the A76 of A-site-bound tRNA, is a psi or is next to a psi in eukaryotes and Archaea, and is five residues away from psi 2580 in E. coli. A2637, which is between the two CCA ends of P- and A-site tRNA, is near psi 2639, psi 2640, and psi 2641, found in a number of organisms. Residue 2529, which contacts the backbone of A-site tRNA residues 74-76, is near psi 2527 psi 2528 in H. marismortui. Residues 2505-2507, which contact A-site tRNA residues 50-53, are near psi 2509 in higher eukaryotes, and residues 2517-2519 in contact with A-site tRNA residues 64-65 are within 1-3 nucleotides of psi 2520 in higher eukaryotes and psi 2514 in H. marismortui. A way to rationalize this might be to invoke the concept suggested in the Introduction that psi acts as a molecular glue to hold loose elements in a more rigid configuration. It may well be that this is more important near the site of peptide-bond formation and tRNA binding, accounting for the preponderance of psi in this vicinity. What might be the role of all the other psi in eukaryotes? One can only surmise that cells, having once acquired the ability to make psi with guide RNAs, took advantage of the system to inexpensively place psi wherever an undesirable loose region was found. It might be that in some of these cases, psi performs the role played by proteins in other regions, namely that of holding the rRNA in its proper configuration. Confirmation of this hypothesis will have to await structural determination of eukaryotic ribosomes.
Asunto(s)
Hidroliasas/metabolismo , Seudouridina/metabolismo , ARN Ribosómico/química , Animales , Bacterias/enzimología , Bacterias/metabolismo , Secuencia de Bases , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Seudouridina/análisis , Ribosomas/enzimología , Ribosomas/metabolismoRESUMEN
All nine pseudouridine (psi) residues in Escherichia coli 23S RNA are in or very near the peptidyl transfer centre (PTC) of the ribosome. Five psi synthases catalyze synthesis of these nine psi's. Deletion of the gene for one psi synthase, RluD, which directs synthesis of three closely clustered psi's in the decoding site of the PTC, has a profound negative impact on cell growth. We describe the isolation, without amplification from a cloned coding element, of the triple-site modifying enzyme, RluD, the N-terminal sequence of which has been used to clone and express the corresponding gene, rluD. Unlike "expressed" RluD, which so far has not been shown to modify one (1911) of the three closely clustered sites (1911, 1915, 1917), "natural" RluD modifies all three sites; and unlike another pai synthase, RluA, natural RluD has greatly expanded modifying activity at low Mg concentrations. These properties of the expressed and natural forms of RluD are discussed.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/enzimología , Hidroliasas , Transferasas Intramoleculares/aislamiento & purificación , Transferasas Intramoleculares/metabolismo , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Transferasas Intramoleculares/química , Magnesio/metabolismo , Datos de Secuencia Molecular , Seudouridina/biosíntesis , ARN Ribosómico 23S/metabolismoRESUMEN
Previous work from this laboratory (Nurse et al., RNA, 1995, 1:102-112) established that TruB, a pseudouridine (psi) synthase from Escherichia coli, was able to make psi55 in tRNA transcripts but not in transcripts of full-length or fragmented 16S or 23S ribosomal RNAs. By deletion of the truB gene, we now show that TruB is the only protein in E. coli able to make psi55 in vivo. Lack of TruB and psi55 did not affect the exponential growth rate but did confer a strong selective disadvantage on the mutant when it was competed against wild-type. The negative selection did not appear to be acting at either the exponential or stationary phase. Transformation with a plasmid vector conferring carbenicillin resistance and growth in carbenicillin markedly increased the selective disadvantage, as did growth at 42 degrees C, and both together were approximately additive such that three cycles of competitive growth sufficed to reduce the mutant strain to approximately 0.2% of its original value. The most striking finding was that all growth effects could be reversed by transformation with a plasmid carrying a truB gene coding for a D48C mutation in TruB. Direct analysis showed that this mutant did not make psi55 under the conditions of the competition experiment. Therefore, the growth defect due to the lack of TruB must be due to the lack of some other function of the protein, possibly an RNA chaperone activity, but not to the absence of psi55.
Asunto(s)
Proteínas Bacterianas/fisiología , Escherichia coli/genética , Liasas Intramoleculares/fisiología , Seudouridina/metabolismo , Secuencia de Aminoácidos , Bacterias/enzimología , Proteínas Bacterianas/genética , Escherichia coli/enzimología , Escherichia coli/crecimiento & desarrollo , Eliminación de Gen , Genes Bacterianos , Prueba de Complementación Genética , Liasas Intramoleculares/deficiencia , Liasas Intramoleculares/genética , Transferasas Intramoleculares , Datos de Secuencia Molecular , Plásmidos/genética , ARN de Transferencia/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Especificidad de la EspecieRESUMEN
The Escherichia coli gene rluA, coding for the pseudouridine synthase RluA that forms 23 S rRNA pseudouridine 746 and tRNA pseudouridine 32, was deleted in strains MG1655 and BL21/DE3. The rluA deletion mutant failed to form either 23 S RNA pseudouridine 746 or tRNA pseudouridine 32. Replacement of rluA in trans on a rescue plasmid restored both pseudouridines. Therefore, RluA is the sole protein responsible for the in vivo formation of 23 S RNA pseudouridine 746 and tRNA pseudouridine 32. Plasmid rescue of both rluA- strains using an rluA gene carrying asparagine or threonine replacements for the highly conserved aspartate 64 demonstrated that neither mutant could form 23 S RNA pseudouridine 746 or tRNA pseudouridine 32 in vivo, showing that this conserved aspartate is essential for enzyme-catalyzed formation of both pseudouridines. In vitro assays using overexpressed wild-type and mutant synthases confirmed that only the wild-type protein was active despite the overexpression of wild-type and mutant synthases in approximately equal amounts. There was no difference in exponential growth rate between wild-type and MG1655(rluA-) either in rich or minimal medium at 24, 37, or 42 degrees C, but when both strains were grown together, a strong selection against the deletion strain was observed.
Asunto(s)
Escherichia coli/genética , Transferasas Intramoleculares/genética , ARN Ribosómico 23S/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/enzimología , Transferasas Intramoleculares/metabolismo , Cinética , Mutagénesis , Seudouridina/genética , ARN Ribosómico 23S/metabolismo , Eliminación de SecuenciaRESUMEN
The gene for RsuA, the pseudouridine synthase that converts U516 to pseudouridine in 16S ribosomal RNA of Escherichia coli, has been deleted in strains MG1655 and BL21/DE3. Deletion of this gene resulted in the specific loss of pseudouridine516 in both cell lines, and replacement of the gene in trans on a plasmid restored the pseudouridine. Therefore, rsuA is the only gene in E. coli with the ability to produce a protein capable of forming pseudouridine516. There was no effect on the growth rate of rsuA- MG1655 either in rich or minimal medium at either 24, 37, or 42 degrees C. Plasmid rescue of the BL21/DE3 rsuA- strain using pET15b containing an rsuA gene with aspartate102 replaced by asparagine or threonine demonstrated that neither mutant was active in vivo. This result supports a role for this aspartate, located in a unique GRLD sequence in this gene, at the catalytic center of the synthase. Induction of wild-type and the two mutant synthases in strain BL21/DE3 from genes in pET15b yielded a strong overexpression of all three proteins in approximately equal amounts showing that the mutations did not affect production of the protein in vivo and thus that the lack of activity was not due to a failure to produce a gene product. Aspartate102 is found in a conserved motif present in many pseudouridine synthases. The conservation and distribution of this motif in nature was assessed.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/genética , Transferasas Intramoleculares/genética , Secuencia de Aminoácidos , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Dominio Catalítico , Escherichia coli/enzimología , Escherichia coli/crecimiento & desarrollo , Eliminación de Gen , Transferasas Intramoleculares/metabolismo , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
RluC from E. coli is the enzyme responsible for catalyzing the isomerization of uridines 955, 2504 and 2580 in 23S rRNA to pseudouridine. Histidine-tagged RluC was cloned, overexpressed and purified by nickel-affinity chromatography. A proteolytically derived fragment of the enzyme consisting of residues 89-319 has been shown to retain catalytic activity. Crystals of this fragment, grown by precipitation with sodium acetate at pH 8.0, belong to space group P321, with unit-cell dimensions a = b = 97.1, c = 86.3 A and have two molecules in the crystallographic asymmetric unit. The flash-frozen crystals diffract X-rays to at least 2.3 A resolution and appear suitable for crystal structure determination.
Asunto(s)
Escherichia coli/enzimología , Hidroliasas/química , Hidroliasas/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Expresión Génica , Hidroliasas/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , ARN Bacteriano , ARN Ribosómico 23S , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The methyltransferase that forms m5C967 in Escherichia coli small subunit ribosomal RNA has been purified, cloned, and characterized. The gene was identified from the N-terminal sequence of the purified enzyme. The gene is a fusion of two open reading frames, fmu and fmv, previously believed to be distinct due to a DNA sequencing error. The gene, here named rsmB, encodes a 429-amino acid protein that has a number of homologues in prokaryotes, Archaea, and eukaryotes. C-Terminal sequencing of the overexpressed and affinity-purified protein by mass spectrometry methods verified the sequence expected for the gene product. The recombinant protein exhibited the same specificity as the previously described native enzyme; that is, it formed only m5C and only at position 967. C1407, which is also m5C in natural 16S RNA, was not methylated. In vitro, the enzyme only recognized free 16S RNA. 30S ribosomal subunits were not a substrate. There was no requirement for added magnesium, suggesting that extensive secondary or tertiary structure in the RNA substrate may not be a requirement for recognition.
Asunto(s)
Citosina/análogos & derivados , Escherichia coli/enzimología , Metiltransferasas/aislamiento & purificación , ARN Ribosómico 16S/química , 5-Metilcitosina , Secuencia de Aminoácidos , Clonación Molecular , Citosina/química , Citosina/metabolismo , Metilación de ADN , Escherichia coli/genética , Expresión Génica , Genes Bacterianos , Magnesio/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Especificidad por SustratoRESUMEN
A Bacillus subtilis ORF, ypul, 41% homologous to rsuA, the gene for the synthase which forms pseudouridine 516 in Escherichia coli 16S rRNA, was cloned and the protein expressed and affinity-purified by the His tag procedure. Reactions with E. coli 16S and 23S rRNA transcripts were performed in vitro. The protein did not form pseudouridine 516 as expected but did produce pseudouridine 552 in 16S rRNA and pseudouridines 1199, 2605, and 2833 in 23S rRNA. Of these, only pseudouridine 2605 is found naturally in either E. coli or B. subtilis rRNA. Kinetic experiments confirmed that pseudouridine 2605 was the primary target. Comparison of the four pseudouridine sites yielded a consensus recognition sequence for the synthase. This consensus sequence was not present at any other site in either E. coli or B. subtilis 16S or 23S RNA. We propose that YpuL is the B. subtilis pseudouridine 2633 (2605 in E. coli) synthase. Since the closest gene sequence homologue in E. coli is yciL, we suggest that its gene product is the corresponding E. coli pseudouridine 2605 synthase.
Asunto(s)
Bacillus subtilis/enzimología , Bacillus subtilis/genética , Transferasas Intramoleculares/genética , ARN Ribosómico 23S/química , Secuencia de Bases , Clonación Molecular , Regulación Bacteriana de la Expresión Génica , Transferasas Intramoleculares/química , Transferasas Intramoleculares/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The methyltransferase that forms m2G1207 in Escherichia coli small subunit rRNA has been purified, cloned, and characterized. The gene was identified from the N-terminal sequence of the purified enzyme as the open reading frame yjjT (SWISS-PROT accession number ). The gene, here renamed rsmC in view of its newly established function, codes for a 343-amino acid protein that has homologs in prokaryotes, Archaea, and possibly also in lower eukaryotes. The enzyme reacted well with 30 S subunits reconstituted from 16 S RNA transcripts and 30 S proteins but was almost inactive with the corresponding free RNA. By hybridization and protection of appropriate segments of 16 S RNA that had been extracted from 30 S subunits methylated by the enzyme, it was shown that of the three naturally occurring m2G residues, only m2G1207 was formed. Whereas close to unit stoichiometry of methylation could be achieved at 0.9 mM Mg2+, both 2 mM EDTA and 6 mM Mg2+ markedly reduced the level of methylation, suggesting that the optimal substrate may be a ribonucleoprotein particle less structured than a 30 S ribosome but more so than free RNA.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/enzimología , Metiltransferasas/aislamiento & purificación , ARN Ribosómico 16S/química , Secuencia de Aminoácidos , Cromatografía de Afinidad , Clonación Molecular , Magnesio/metabolismo , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta , Especificidad por SustratoRESUMEN
Escherichia coli rRNA contains 10 pseudouridines of unknown function. They are made by synthases, each of which is specific for one or more pseudouridines. Here we show that the sfhB (yfil) ORF of E. coli is a pseudouridine synthase gene by cloning, protein overexpression, and reaction in vitro with rRNA transcripts. Gene disruption by miniTn10(cam) insertion revealed that this synthase gene, here renamed rluD, codes for a synthase which is solely responsible in vivo for synthesis of the three pseudouridines clustered in a stem-loop at positions 1911, 1915, and 1917 of 23S RNA. The absence of RluD results in severe growth inhibition. Both the absence of pseudouridine and the growth defect could be reversed by insertion of a plasmid carrying the rluD gene into the mutant cell, clearly linking both effects to the absence of RIuD. This is the first report of a major physiological defect due to the deletion of any pseudouridine synthase. Growth inhibition may be due to the lack of one or more of the 23S RNA pseudouridines made by this synthase since pseudouridines 1915 and 1917 are universally conserved and are located in proximity to the decoding center of the ribosome where they could be involved in modulating codon recognition.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos/genética , Hidroliasas , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Seudouridina/biosíntesis , ARN Ribosómico 23S/genética , Secuencia de Aminoácidos , Secuencia de Bases , División Celular , Clonación Molecular , Escherichia coli/crecimiento & desarrollo , Genes Esenciales/genética , Prueba de Complementación Genética , Transferasas Intramoleculares/aislamiento & purificación , Datos de Secuencia Molecular , Mutagénesis Insercional , Conformación de Ácido Nucleico , Fenotipo , Reacción en Cadena de la Polimerasa , ARN Bacteriano/biosíntesis , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Ribosómico 23S/biosíntesis , ARN Ribosómico 23S/química , Análisis de Secuencia de ADN , Análisis de Secuencia de ARNRESUMEN
Escherichia coli ribosomal RNA contains 10 pseudouridines, one in the 16 S RNA and nine in the 23 S RNA. Previously, the gene for the synthase responsible for the 16 S RNA pseudouridine was identified and cloned, as was a gene for a synthase that makes a single pseudouridine in 23 S RNA. The yceC open reading frame of E. coli is one of a set of genes homologous to these previously identified ribosomal RNA pseudouridine synthases. In this work, the gene was cloned, overexpressed, and shown to code for a pseudouridine synthase able to react with in vitro transcripts of 23 S ribosomal RNA. Deletion of the gene and analysis of the 23 S RNA from the deletion strain for the presence of pseudouridine at its nine known sites revealed that this synthase is solely responsible in vivo for the synthesis of three of the nine pseudouridine residues, at positions 955, 2504, and 2580. Therefore, this gene has been renamed rluC. Despite the absence of one-third of the normal complement of pseudouridines, there was no change in the exponential growth rate in either LB or M-9 medium at temperatures ranging from 24 to 42 degrees C. From this work and our previous studies, we have now identified three synthases that account for 50% of the pseudouridines in the E. coli ribosome.
Asunto(s)
Escherichia coli/genética , Transferasas Intramoleculares/genética , Seudouridina/biosíntesis , ARN Ribosómico 23S/metabolismo , Secuencia de Bases , Escherichia coli/enzimología , Eliminación de Gen , Glucosa/metabolismo , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sistemas de Lectura AbiertaRESUMEN
Biochemical and genetic studies have pointed out the importance of several sites in 16S ribosomal RNA of Escherichia coli in the decoding process. These sites consist of the core of the decoding center (1400/1500 region) and two other segments (530 and 1050/1200 regions). To detect a possible structural link between these functionally related regions, we analyzed their sensitivity to conformational changes induced by mutations which are located in each of these regions and are known to affect the decoding process. The conformations of five segments of 16S rRNA (1-106, 406-569, 780-978, 997-1247, and 1334-1519) were analyzed by chemical probing of 30S ribosomes containing the following mutations: G530U, U1498G, G1401C, C1501G, and G1401C/C1501G. Ribosomes reconstituted with natural wild-type 16S RNA showed only minor conformational differences with respect to ribosomes isolated from cells. When 16S RNA made in vitro replaced natural 16S RNA, a slightly looser conformation of the central core region was found. Mutant ribosomes made by reconstitution with mutant 16S RNA made in vitro showed conformational effects which were in all cases localized to the region of secondary structure surrounding the site of mutation. Although the core of the decoding center (1400/1500 region) and the two other sites (530 and 1050/1200 regions) participating in the decoding function have been functionally linked, our data indicate that they are structurally independent. They also provide evidence for an unusual structure of the 1400/1500 decoding center, possibly involving noncanonical interactions. Furthermore, the absence of any conformational effect induced by the G530U mutation except at the site of mutation itself points to its direct, as opposed to indirect, involvement in the decoding function of the ribosome.
Asunto(s)
Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Ribosómico 16S/química , Secuencia de Bases , Nucleótidos de Citosina/genética , Desoxiuridina , Escherichia coli/genética , Nucleótidos de Guanina/genética , Datos de Secuencia Molecular , ARN Bacteriano/genética , ARN Ribosómico 16S/síntesis química , ARN Ribosómico 16S/genética , Ribosomas/química , Ribosomas/genética , Relación Estructura-ActividadRESUMEN
The pseudouridine (psi) residues present in the high molecular mass RNA from the large ribosomal subunit (LSU) have been sequenced from representative species of the eukaryotes, prokaryotes and archaebacteria, and from mitochondrial and chloroplast organelles. Ribosomes from Bacillus subtilis, Halobacter halobium, Drosphilia melanogaster, Mus musculus, Homo sapiens, mitochondria of M. musculus, H. sapiens and Trypanosoma brucei, and Zea mays chloroplasts were examined, resulting in the exact localization of 190 psi residues. The number of psi residues per RNA varied from one in the mitochondrial RNAs to 57 in the cytoplasmic LSU RNA of D. melanogaster and M. musculus. Despite this, all of the psi residues were found in three domains, II, IV and V. All three are at or have been linked to the peptidyl transferase center according to the literature. Comparison of the sites for psi among the species examined revealed four conserved or semi-conserved segments. One is the region 1911 to 1917, which contains three psi or modified psi in almost all species examined. This site is also juxtaposed to the decoding site of the 30 S subunit in the 70 S ribosome and has been implicated in the fidelity of codon recognition. Three additional sites were at the peptidyl transferase center itself. The juxtaposition of the conserved sites for psi with the two important functions of the ribosome, codon recognition and peptide bond formation, implies an important role for psi in ribosome function. We report some new putative modified nucleosides in LSU RNAs as detected by reverse transcription, correct a segment of the sequence of Z. mays chloroplasts and D. melanogaster LSU RNA, correlate the secondary structural context for all known psi residues in ribosomal RNA, and compare the sites for psi with those known for methylated nucleosides in H. sapiens.
Asunto(s)
Cloroplastos/genética , Halobacterium salinarum/genética , Mitocondrias/genética , Seudouridina/análisis , ARN Ribosómico/química , Animales , Bacillus subtilis/genética , Secuencia de Bases , Sitios de Unión , Drosophila melanogaster/genética , Humanos , Ratones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Nucleósidos/química , Nucleósidos/genética , Peptidil Transferasas/genética , Seudouridina/química , Seudouridina/genética , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Trypanosoma brucei brucei/genética , Zea mays/genéticaRESUMEN
Mass spectrometry-based methods have been used to study post-transcriptional modification in the 1900-1974 nt segment of domain IV in 23S rRNA of Escherichia coli, a region which interacts with domain V in forming the three- dimensional structure of the peptidyl transferase center within the ribosome. A nucleoside constituent of M r 258 (U*)which occurs at position 1915, within the highly modified oligonucleotide sequence 1911-psiAACU*Apsi-1917, was characterized as 3-methylpseudouridine (m3psi). The assignment was confirmed by chemical synthesis of m3psi and comparison with the natural nucleoside by liquid chromatography-mass spectrometry. 3-Methylpseudouridine is previously unknown in nature and is the only known derivative of the common modified nucleoside pseudouridine thus far found in bacterial rRNA.
Asunto(s)
Escherichia coli/química , Seudouridina/análogos & derivados , ARN Ribosómico 23S/química , Secuencia de Bases , Espectrometría de Masas , Datos de Secuencia Molecular , Estructura Molecular , Seudouridina/químicaRESUMEN
Pseudouridine (psi), the most common single modified nucleoside in ribosomal RNA, has been positioned in the small subunit (SSU) and large subunit (LSU) RNAs of a number of representative species. Most of the information has been obtained by application of a rapid primed reverse transcriptase sequencing technique. The locations of these psi residues have been compared. Many sites for psi are the same among species, but others are distinct. In general, the percentage psi in multicellular eukaryotes is greater than in prokaryotes. In LSU RNA, the psi residues are strongly clustered in three domains, all of which are near or connected to the peptidyl transferase center. There is no apparent clustering of psi in SSU RNA. The psi sites in LSU RNA overlap those for the methylated nucleosides, but this is not the case in SSU RNA. There are 265 psi sites known to nucleotide resolution, of which 246 are in defined secondary structures, and 112 of these are in nonidentical structural contexts. All 246 psi sites can be classified into five structural types. Two Escherichia coli psi synthases have been cloned and characterized, one for psi 516 in SSU RNA and one for psi 746 in LSU RNA. The psi 746 synthase recognizes free RNA, but the psi 516 enzyme requires an intermediate RNP particle. Possible functional roles for psi in the ribosome are discussed.