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PubMed1994

The importance of conserved nucleotides of 23 S ribosomal RNA and transfer RNA in ribosome catalyzed peptide bond formation.

We have constructed the double mutant G2252C/G2253C in Escherichia coli 23 S rRNA by site-directed mutagenesis. These phylogenetically conserved residues are protected from chemical modification by the 3' CCA terminus of the peptidyl-tRNA site (P site)-bound tRNA. Expression of C2252/C2253 23 S rRNA in E. coli severely compromises cell growth. Mutant rRNA is assembled into 50 S subunits and 70 S ribosomes but is discriminated against in polysomes. Mutant ribosomes function at lower rates in peptidyltransferase assays than wild type ribosomes. To test whether this defect derives from disruption of base pairing with the 2 cytidines of the invariant 3' CCA terminus of tRNA, a mutant E. coli tRNAPhe gene was constructed, with the CCA sequence changed to GGA. As deacylated species, mutant and wild type tRNAPhe inhibit peptidyl transfer identically. Mutant tRNAPhe was aminoacylated in vitro but failed to react as a P site substrate, with either mutant or wild type ribosomes. These results support a role for G2252 and G2253 of 23 S rRNA in peptidyltransferase function and a role for the 3' residues of peptidyl-tRNA in catalytically productive P site interaction; but they fail to provide evidence supporting canonical base pairing between these 23 S residues and the 3' end of peptidyl-tRNA.

Base Sequence↗
PubMed1983

The role of queuine in the aminoacylation of mammalian aspartate transfer RNAs.

Can a queuine-specific tRNA function normally without replacement of G by Q in its structure? To answer this, kinetics of aspartate queuine-containing tRNA (Q-tRNA) is compared with its queuine-deficient counterpart (G-tRNA). The results indicate that Asp Q-tRNA is a more effective substrate than the Asp G-tRNA. The Asp Q-tRNA exhibits a higher reaction velocity (Vmax greater than 30%) and a higher reaction rate (Km less than 55%) than its counterpart. The Asp tRNAs derived from human tumor lines and grown in athymic mice contain a full complement of queuine. This tumor tRNA exhibits aminoacylation kinetics similar to a normal liver tRNA. Reasons for observing the lack of a G-to-Q modification in cancer tRNAs by others are hypothesized. Two purified Asp isoacceptors from liver are compared for the aminoacylation reaction; small differences are noted in the Vmax, but none in the Km values.

Animals↗
PubMed1998

Identification by modification-interference of purine N-7 and ribose 2'-OH groups critical for catalysis by bacterial ribonuclease P.

The RNA subunit of bacterial ribonuclease P is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. A self-cleaving RNase P RNA-substrate conjugate was used in modification-interference analysis to identify purine N-7 and ribose 2'-hydroxyl functional groups that are critical to catalysis. We identify six adenine N-7 groups and only one 2'-hydroxyl that, when substituted with 7-deazaadenine or 2'-deoxy analogues, respectively, reduce the RNase P catalytic rate approximately 10-fold at pH 8 and limiting concentration of magnesium. Two sites of low-level interference by phosphorothioate modification were detected in addition to the four sites of strong interference documented previously. These modification-interference results, the absolute phylogenetic conservation of these functional groups in bacterial RNase P RNA, their proximity to the substrate-phosphate in the tertiary structure of the ribozyme-substrate complex, and the importance of some of the sites for binding of catalytic magnesium all implicate these functional groups as components of the RNase P active site. Five of the 7-deazaadenine interferences are suppressed at pH 6, where the hydrolytic step is rate-limiting, or at saturating concentrations of magnesium. We propose, therefore, that these base functional groups are specifically engaged in the catalytic center of RNase P RNA, possibly by involvement in magnesium-dependent folding. One 7-deazaadenine interference and one 2'-deoxy-interference, although partially suppressed at pH 6, are not suppressed at saturating magnesium concentrations. This implicates these groups in magnesium-independent folding of the catalytic substructure of the ribozyme.

Base Sequence↗
PubMed1990

Recognition of tRNA identity determinants by aminoacyl-tRNA synthetases.

By analyzing aminoacylation activities of variants of isoleucine tRNAs and glutamic acid tRNA from Escherichia coli, it was found that the anticodons are the major determinants for "identities" of these tRNAs. It was also shown that the post-transcriptional modifications are essential to aminoacylation of these tRNAs. Interactions between the tRNAs and the aminoacyl-tRNA synthetases were analyzed by NMR spectroscopy.

Amino Acyl-tRNA Synthetases↗
PubMed1999

Role of metal ions in the hydrolysis reaction catalyzed by RNase P RNA from Bacillus subtilis.

Precursor tRNA (ptRNA) substrates carrying a single Rp or Sp-phosphorothioate modification at the RNase P cleavage site were used as tools to study the cleavage mechanism of RNase P RNA from Bacillus subtilis. Both the Sp and the Rp-diastereomer reduced the rate of processing at least 10(4)-fold under conditions where the chemical step is essentially rate-limiting. Neither the Rp nor the Sp-phosphorothioate modification affected ptRNA ground state binding to B. subtilis RNase P RNA. Processing of the Rp-diastereomeric ptRNA could be restored in the presence of Mn2+or Cd2+, demonstrating direct metal ion coordination to the pro -Rp oxygen during catalysis. With Cd2+, processing required the presence of another metal ion, such as Ca2+or Mg2+, to mediate substrate binding. This is in contrast to Escherichia coli RNase P RNA, which promotes cleavage of Rp-diastereomeric ptRNA in the presence of Cd2+as the sole divalent metal ion. Analysis of [Cd2+]-dependent processing of the Rp-diastereomeric substrate by B. subtilis RNase P RNA was consistent with the involvement of at least two metal ions in catalysis. The presence of two catalytic metal ion binding sites is also supported by the inhibition mode of Ca2+on cleavage of unmodified ptRNA. In the presence of an Sp-phosphorothioate modification at the scissile bond, neither Mn2+nor Cd2+were able to restore significant cleavage at this location. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Unaffected ground state binding of the Sp-diastereomeric ptRNA but a >/=10(4)-fold reduced hydrolysis rate may indicate a crucial role of the pro -Sp oxygen in transition state stabilization or may be attributed to steric exclusion of catalytic metal ions. Based on our comparative analyses of B. subtilis and E. coli RNase P RNA, each representing the main structural subtypes of bacterial RNase P RNA, common features in terms of active site constraints and role of catalytic metal ions can now be formulated for bacterial RNase P RNAs. On the other hand, substantial and unexpected differences with respect to the overall metal ion requirements and tRNA binding modes have been observed for the two catalytic RNAs.

Bacillus subtilis↗
PubMed1992

Chloroplast tRNA(Asp): nucleotide sequence and variation of in vivo levels during plastid maturation.

Two chloroplast tRNA(Asp) species from barely were purified by chromatography on benzoylated DEAE-cellulose and sequenced. They differ in the modification at position 34, where queuosine (Q) is present in one of the species. The same chromatographic procedure yielded only one tRNA(Glu) species, corroborating the assumption that the same tRNA(Glu) species participates in both protein and chlorophyll biosynthesis. The level of tRNA(Glu) remains unchanged after light treatment of etiolated seedlings, whereas the amount of tRNA(Asp) decreases to about 50% relative to the level of dark-grown plants.

Base Sequence↗
PubMed2025

The Biosynthetic Pathway to the Pyrroloiminoquinone Marine Natural Product Ammosamide C.

Ammosamide C is a marine natural product containing a highly decorated pyrroloiminoquinone core. Studies on the biosynthetic gene cluster (BGC) that produces ammosamides previously revealed that they are made by a series of posttranslational modifications (PTMs). The BGC includes genes encoding a precursor peptide AmmA and four enzymes known as PEptide Aminoacyl-tRNA Ligases (PEARLs). Initial studies into the ammosamide biosynthetic pathway demonstrated Trp addition to a precursor peptide by the PEARL AmmB2. Thereafter, sequential modifications by several enzymes, including two other PEARLs lead to the formation of a peptide intermediate bearing a C-terminal diaminoquinone. In the present work, we present the biosynthetic steps that convert this intermediate to ammosamide C. The PEARL AmmB4 unexpectedly appends an arginine to the C-terminus of the aforementioned intermediate. Then, C-terminal proteolysis by the heterodimeric TldD/E-like protease Amm12/13 releases a dipeptide, which is subsequently cleaved by the dipeptidase Amm19 to produce a Trp-derived diaminoquinone. Amm3 next catalyzes the conversion of this Trp derivative to the corresponding chlorinated ammosamaic acid. Finally, a putative aminotransferase Amm20 performs an amidation, and Amm23 methylates this intermediate to arrive at ammosamide C; the order of these last two steps could not be determined definitively. This study reveals an unexpectedly lengthy route to ammosamide that illustrates the opportunistic nature of natural product biosynthesis, demonstrates a role for a PEARL that is unlike previous roles, identifies steps that are not PTMs, and adds Arg-tRNA to the growing repertoire of aminoacyl tRNAs that are used by PEARLs.

Biological Products↗
PubMed1970

Formation of all stable RNA species in Escherichia coli by posttranscriptional modification.

The kinetics of accumulation of the known stable RNA species (23S, 16S, and 5S rRNA and tRNA) in Escherichia coli C122 were monitored by polyacrylamide gel electrophoresis of purified cellular RNA, following termination of brief pulse labeling with (32)P-orthophosphate. Isotopically labeled stable RNA species appear only after a time lag, while total cellular RNA and the ostensible precursors to the stable RNA classes accumulate from the earliest times examined. It is concluded that all the known stable RNA species in E. coli are the products of posttranscriptional modification.

Electrophoresis, Disc↗
PubMed1982

Macromolecular complexes from sheep and rabbit containing seven aminoacyl-tRNA synthetases. II. Structural characterization of the polypeptide components and immunological identification of the methionyl-tRNA synthetase subunit.

The extensively purified multienzyme complexes from sheep and rabbit livers containing seven aminoacyl-tRNA synthetases specific for Ile, Leu, Met, Gln, Glu, Lys, and Arg displayed characteristic one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic patterns composed of 11 and 10 major polypeptide components, respectively. Their polypeptide compositions revealed by two-dimensional electrophoresis, including isoelectric focusing in 9 M urea, were not significantly more complex. The isoelectric point of each component from the two complexes fell within the pH range of 6.2 to 7.1, with the notable exception of the common polypeptide of Mr = 43,000 which was distinctly basic. The apparent molecular weight of each component from both complexes was determined by SDS-polyacrylamide gel electrophoresis. Four polypeptides, corresponding to molecular weights of 139,000, 129,000, 43,000, and 38,000 were common to both complexes. The other components from the two complexes displayed similar yet clearly distinct molecular weights. The molar ratios of the polypeptides, estimated by densitometry scanning of stained SDS-polyacrylamide gels, indicated that several components from each complex may be present as more than one copy. Following SDS-polyacrylamide gel electrophoresis, the methionyl-tRNA synthetase component from each complex was identified by the protein blotting procedure, using specific antibodies and 125I-labeled protein A. The unique labeled bands from the complexes of sheep and rabbit precisely matched the major polypeptides of Mr = 103,000 and 108,000, respectively. Mild trypsin treatment of the two native complexes generated fully active forms of methionyl-tRNA synthetase, with molecular weights of 68,000 and 69,500, respectively. The kinetics of proteolysis showed that modification proceeded sequentially through discrete intermediates.

Amino Acyl-tRNA Synthetases↗
PubMed2001

Chemical accessibility of 18S rRNA in native ribosomal complexes: interaction sites of mRNA, tRNA and translation factors.

During protein synthesis the ribosome interacts with ligands such as mRNA, tRNA and translation factors. We have studied the effect of ribosome-ligand interaction on the accessibility of 18S rRNA for single strand-specific modification in ribosomal complexes that have been assembled in vivo, i. e. native polysomes. A comparison of the modification patterns derived from programmed and non-programmed ribosomes showed that bases in the 630- and 1060-loops (530- and 790-loops in E. coli) together with two nucleotides in helices 33 and 34 were protected from chemical modification. The majority of the protected sites were homologous to sites previously suggested to be involved in mRNA and/or tRNA binding in prokaryotes and eukaryotes, implying that the interaction sites for these ligands are similar, if not identical, in naturally occurring programmed ribosomes and in in vitro assembled ribosomal complexes. Additional differences between programmed and non-programmed ribosomes were found in hairpin 8. The bases in helix 8 showed increased exposure to chemical modification in the programmed ribosomes. In addition, structural differences in helices 36 and 37 were observed between native 80S run-off ribosomes and 80S ribosomes assembled from isolated 40S and 60S subunits.

Animals↗
PubMed2005

Introduction of hypermodified nucleotides in RNA.

The anticodon domain of lysine transfer ribonucleic acid (tRNA) is a model system for investigation of the structural and biochemical effects of nucleoside posttranscriptional modification. To enable detailed study of the biophysical and structural effects of hypermodified nucleosides, methods have been developed to synthesize RNA oligonucleotides containing the modified nucleosides found in lysine tRNA. We describe in detail the synthesis of protected phosphoramidites of the nucleosides methylaminomethyl-2-thiouridine (mnm5s2U), methylcarboxymethyl-2-thiouridine (mcm5s2U), and 2-thiomethyl-N-6-carbamoylthreonyl-adenosine (ms2t6A). We also describe methods for using these nucleoside phosphoramidite reagents to synthesize RNA oligonucleotides with modified nucleosides incorporated at the specific sequence locations corresponding to their positions in the native lysine tRNAs.

Nucleotides↗
PubMed2001

Post-transcriptional modification in archaeal tRNAs: identities and phylogenetic relations of nucleotides from mesophilic and hyperthermophilic Methanococcales.

Post-transcriptional modifications in archaeal RNA are known to be phylogenetically distinct but relatively little is known of tRNA from the Methanococci, a lineage of methanogenic marine euryarchaea that grow over an unusually broad temperature range. Transfer RNAs from Methanococcus vannielii, Methanococcus maripaludis, the thermophile Methanococcus thermolithotrophicus, and hyperthermophiles Methanococcus jannaschii and Methanococcus igneus were studied to determine whether modification patterns reflect the close phylogenetic relationships inferred from small ribosomal subunit RNA sequences, and to examine modification differences associated with temperature of growth. Twenty-four modified nucleosides were characterized, including the complex tricyclic nucleoside wyosine characteristic of position 37 in tRNA(Phe) and known previously only in eukarya, plus two new wye family members of presently unknown structure. The hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, reported previously only in bacterial tRNA at the first position of the anticodon, was identified by liquid chromatography-electrospray ionization mass spectrometry in four of the five organisms. The ribose-methylated nucleosides, 2'-O-methyladenosine, N(2),2'-O-dimethylguanosine and N(2),N(2),2'-O-trimethylguanosine, were found only in hyperthermophile tRNA, consistent with their proposed roles in thermal stabilization of tRNA.

Chromatography, High Pressure Liquid↗
PubMed1997

GTP-dependent modification of a 21-kDa substrate with NAD+ in bovine brain soluble fraction is not ADP-ribosylation of small G-protein but tailing of tRNA.

Labeling of 21-kDa material was observed when bovine brain soluble fraction was incubated with [adenylate-32P]NAD+ in the presence of GTP. The 21-kDa substrate, slightly smaller than C3 substrate in size, was labeled even without C3 exoenzyme. GTP could be replaced by nucleoside triphosphates other than ATP while ATP inhibited the GTP-induced labeling of 21-kDa substrate. After incubation of the soluble fraction with [adenylate-32P]NAD+ in the presence of GTP, [32P]ADP and [32P]ATP were detected in addition to [32P]AMP and [32P]ADP-ribose while only the last two nucleotides were observed without GTP. The 21-kDa substrate was labeled with [alpha-32P]ATP even in the absence of GTP, suggesting adenylylation rather than ADP-ribosylation. The labeled 21-kDa substrate, was extractable by phenol, disappeared with RNase treatment but not with tryptic digestion. Alkaline treatment of the phenol extract yielded an equal mixture of 3'-[32P]CMP and 2'-[32P]CMP. From these results we concluded that the 21-kDa labeling is a result of tRNA tailing with [alpha-32P]ATP generated from the [32P]AMP moiety of [adenylate-32P]NAD+. Results from reconstitution experiments using enzymes and tRNA purified from bovine brain soluble fraction, which are involved in this pathway, confirmed our conclusion.

ADP Ribose Transferases↗
PubMed1994

tRNA-guanine transglycosylase from Escherichia coli: recognition of dimeric, unmodified tRNA(Tyr).

In order to probe the interaction between tRNA and the tRNA hypermodifying enzyme, tRNA-guanine transglycosylase (TGT) from Escherichia coli, we have undertaken the generation of E coli tRNA(Tyr) and analogues. During efforts to adapt currently available in vitro transcription techniques we encountered difficulties attributable to dimerization of the tRNA products. E coli tRNA(Tyr) has previously been characterized for its ability to form a dimer in solutions of suitable salt concentrations at appropriate temperatures (Yang SK, Söll DG, Crothers DM (1972) Biochemistry 11, 2311-2320; Rordorff BF, Kearns DR (1976) Biochemistry 15, 3320-3330). We have applied similar techniques to our unmodified analogue of E coli tRNA(Tyr) and produced both monomeric and dimeric forms of E coli tRNA(Tyr). In this report we find that the dimer does serve as a substrate for modification by TGT. While both the conformers are equal in terms of Vmax (within experimental error) a 2.5-fold increase in KM occurs when going from monomer to dimer. This suggests that TGT preferentially binds the monomer but once either conformer is bound will catalyze the modification reaction equally well. We have also compared the results for the two conformers to our previous data of an RNA minihelix corresponding to the anticodon arm of E coli tRNA(Tyr). Here we find that our earlier conclusion, that the recognition elements for TGT are localized within the anticodon arm of cognate tRNAs, is supported.

Base Sequence↗
PubMed1999

Mutations in the conserved P loop perturb the conformation of two structural elements in the peptidyl transferase center of 23 S ribosomal RNA.

Evidence is presented for the participation of the P loop (nucleotides G2250-C2254) of 23 S rRNA in establishing the tertiary structure of the peptidyl transferase center. Single base substitutions were introduced into the P loop, which participates in peptide bond formation through direct interaction with the CCA end of P site-bound tRNA. These mutations altered the pattern of reactivity of RNA to chemical probes in a structural subdomain encompassing the P loop and extending roughly from G2238 to A2433. Most of the effects on chemical modification in the P loop subdomain occurred near sites of tertiary interactions inferred from comparative sequence analysis, indicating that these mutations perturb the tertiary structure of this region of RNA. Changes in chemical modification were also seen in a subdomain composed of the 2530 loop (nucleotides G2529-A2534) and the A loop (nucleotides U2552-C2556), the latter a site of interaction with the CCA end of A site-bound tRNA. Mutations in the P loop induced effects on chemical modification that were commensurate with the severity of their characterized functional defects in peptide bond formation, tRNA binding and translational fidelity. These results indicate that, in addition to its direct role in peptide bond formation, the P loop contributes to the tertiary structure of the peptidyl transferase center and influences the conformation of both the acceptor and peptidyl tRNA binding sites.

Base Sequence↗
PubMed1974

Studies on chemical modification of thionucleosides in the transfer ribonucleic acid of Escherichia coli.

(35)S-labelled tRNA from Escherichia coli was treated with chemical reagents such as CNBr, H(2)O(2), NH(2)OH, I(2), HNO(2), KMnO(4) and NaIO(4), under mild conditions where the four major bases were not affected. Gel filtration of the treated tRNA showed desulphurization to various extents, depending on the nature of the reagent. The treated samples after conversion into nucleosides were chromatographed on a phosphocellulose column. NH(2)OH, I(2) and NaIO(4) reacted with all the four thionucleosides of E. coli tRNA, 4-thiouridine (s(4)U), 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U), 2-thiocytidine (s(2)C) and 2-methylthio-N(6)-isopentenyladenosine (ms(2)i(6)A), to various extents. CNBr, HNO(2) and NaHSO(3) reacted with s(4)U, mnm(5)s(2)U and s(2)C, but not with ms(2)i(6)A. KMnO(4) and H(2)O(2) were also found to react extensively with thionucleosides in tRNA. Iodine oxidation of (35)S-labelled tRNA showed that only 6% of the sulphur was involved in disulphide formation. Desulphurization of E. coli tRNA with CNBr resulted in marked loss of acceptor activities for glutamic acid, glutamine and lysine. Acceptor activities for alanine, arginine, glycine, isoleucine, methionine, phenylalanine, serine, tyrosine and valine were also affected, but to a lesser extent. Five other amino acids tested were almost unaffected. These results indicate the fate of thionucleosides in tRNA when subjected to various chemical reactions and the involvement of sulphur in aminoacyl-tRNA synthetase recognition of some tRNA species of E. coli.

Chromatography, Gel↗
PubMed1998

Conformational transitions of an unmodified tRNA: implications for RNA folding.

Unmodified tRNAs are powerful systems to study the effects of posttranscriptional modifications and site-directed mutations on both the structure and function of these ribonucleic acids. To define the general limitations of synthetic constructs as models for native tRNAs, it is necessary to elucidate the conformational states of unmodified tRNAs as a function of solution conditions. Here we report the conformational properties of unmodified yeast tRNAPhe as a function of ionic strength, [Mg2+], and temperature using a combination of spectroscopic measurements along with chemical and enzymatic probes. We find that in low [Na+] buffer at low temperature, native yeast tRNAPhe adopts tertiary structure in the absence of Mg2+. By contrast, tertiary folding of unmodified yeast tRNAPhe has an absolute requirement for Mg2+. Below the melting temperature of the cloverleaf, unmodified yeast tRNAPhe exists in a Mg2+-dependent equilibrium between secondary and tertiary structure. Taken together, our findings suggest that although the tertiary structures of tRNAs are broadly comparable, the intrinsic stability of the tertiary fold, the conformational properties of intermediate states, and the stability of intermediate states can differ significantly between tRNA sequences. Thus, the use of unmodified tRNAs as models for native constructs can have significant limitations. Broad conclusions regarding "tRNA folding" as a whole must be viewed cautiously, particularly in cases where structural changes occur, such as during protein synthesis.

Base Sequence↗
PubMed1981

Photocrosslinking of thiolated aminoacyl-tRNA to ribosomal RNA and proteins.

tRNA has been converted to a form that can be photoactivated by chemical modification of some of the exposed cytidine residues to thio-4-uridine A certain percentage of the modified molecules can be charged and bound to the ribosome; thiolated fMet-tRNAfMet is bound to the P-site as shown by puromycin reactivity. Near the UV irradiation produces covalent crosslinks between total thiolated AA-tRNA or fMet-tRNAfMet and the ribosome. AA-tRNA becomes crosslinked to both 30S and 50S subunits but fMet-tRNAfMet to 50S subunits alone. In each case, crosslinking of tRNA was found to be not only to ribosomal proteins, but also to rRNA. The covalent complexes appear sufficiently stable to allow identification of the proteins or rRNA sequences involved.

Escherichia coli↗