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Methionyl-tRNA synthetase of Escherichia coli. A zinc metalloprotein.

The native dimeric form of methionyl-tRNA synthetase of Escherichia coli contains two zinc atoms per dimer, one per subunit. The bound zinc is retained upon trypsin modification which yields a monomer with one zinc atom. The enzymatic activity of both the dimeric forms is reversibly inhibited by 1,10-phenanthroline but not by its non-chelating analogues. In addition, the native enzyme binds two Mn2+ per dimer with a binding constant of approx. 70 micron but no binding is observed with the trypsin-modified monomer.

Amino Acyl-tRNA Synthetases↗

Effects of post-transcriptional base modifications on the site-specific function of transfer RNA in eukaryote translation.

The site-specific function in translation of several naturally occurring mammalian transfer RNAs has been studied in a series of investigations with some similarities to studies in other laboratories of tRNAs in suppression. Equal amounts of aminoacyl-tRNA isoacceptors with contrasting isotopes were added in pairs to reticulocyte lysates and allowed to incorporate their amino acids into rabbit globin. Rates of incorporation from unlimiting amounts of each isoacceptor into the corresponding amino-acid-containing sites were determined. The tRNAs of each isoacceptor pair differed as to post-transcriptional base modifications. The natural occurrence of these isoacceptors can be correlated with rates of cellular division, with more rapidly dividing and neoplastic cells containing hypomodified tRNA. The overall incorporation of lysine into globin from a fully modified tRNALys that decodes AAG is faster by 25 to 30% than from the corresponding hypomodified tRNALys. There is considerable scatter in values for incorporation ratios at different lysine-containing sites, with the hypomodified isoacceptor even being preferred at one site. The AAG decoding isoacceptors are capable of translating AAA although much more slowly than AAG. In translating AAA, in contrast to translating AAG, the hypomodified tRNALys isoacceptor is preferred. A Y base-deficient hypomodified tRNAPhe isoacceptor found only in some kinds of rapidly dividing tumor cells donates its phenylalanine preferentially to globin in competition with the fully modified Y-containing tRNAPhe of liver by 15 to 17%. There is a considerable range of incorporation ratios at the different phenylalanine-containing sites of the globin subunits. No correlation can be made between the isoacceptor preferred and the phenylalanine codon being translated. The incorporation of histidine from a fully modified tRNAHis-containing Q base in its anticodon, compared with that from the hypomodified counterpart isoacceptor that lacks Q base and that occurs in rapidly dividing cells, showed no difference in their ability to incorporate overall or into individual histidine-containing sites. There is little evidence that adjacent bases or codons in messenger RNA affect the tRNAs preferred in the translation of most sites. A striking pattern of tRNA preference was observed in three cases in which there are tandem codons, with the same codon appearing twice in succession.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

Yeast tRNA-splicing endonuclease cleaves precursor tRNA in a random pathway.

Introns interrupt many of the tRNA genes of Saccharomyces cerevisiae at a constant position in the anticodon loop. Pre-tRNA transcripts must be accurately cleaved at 3' and 5' splice sites by tRNA endonuclease to release these introns. In order to study splice site cleavage order, substrates were prepared in which the ribose 2'-OH at each of the splice sites was phosphorylated. This modification blocked cleavage by the endonuclease. We found that whichever splice site was blocked the endonuclease can cleave the other site, indicating that the two splice sites were cleaved independently. The endonuclease also cleaved both 3'- and 5'-nicked pre-tRNA(Phe). In addition, both kinds of "2/3 molecules" (exon+intron) were observed in kinetic studies, indicating that they were true biochemical intermediates. The rates of cleavage at the 3' and 5' splice sites of pre-tRNA were compared in several ways. The results showed that the endonuclease cleaves 3' and 5' sites at almost the same rate in the first cleavage, whereas in the second cleavage the 3' site was cleaved faster, indicating that the rates of the two routes for cleavage were unequal. These results demonstrated that the endonuclease cleaved pre-tRNA in a random order, creating two routes for removal of introns from pre-tRNA.

Bacteriophage T7↗

Sequence and structure of a serine transfer RNA with GCU anticodon from mosquito mitochondria.

We have determined the primary sequence and modification status of a transfer RNA from mosquito mitochondria whose GCU anticodon indicates that it is a serine tRNA (tRNASerGCU), and have obtained information on higher order structure using partial digestion with nucleases S1 and T1 under non-denaturing conditions. Although its primary sequence homology to mammalian mitochondrial tRNASerGCU is modest (46%), the mosquito tRNA resembles its mammalian mitochondrial counterpart in that a plausible secondary structure configuration includes a drastically abbreviated D arm and a sex base-pair anticodon stem. Other unusual features include a ribose-methylated cytidine residue at the end of the anticodon stem, and the likely occurrence of a psi residue between the amino acid arm and arm IV.

Aedes↗

MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli.

Thionucleosides are uniquely present in tRNA. In many organisms, tRNA specific for Lys, Glu, and Gln contain hypermodified 2-thiouridine (s(2)U) derivatives at wobble position 34. The s(2) group of s(2)U34 stabilizes anticodon structure, confers ribosome binding ability to tRNA and improves reading frame maintenance. Earlier studies have mapped and later identified the mnmA gene (formerly asuE or trmU) as required for the s(2)U modification in Escherichia coli. We have prepared a nonpolar deletion of the mnmA gene and show that it is not required for viability in E. coli. We also cloned mnmA from E. coli, and overproduced and purified the protein. Using a gel mobility shift assay, we show that MnmA binds to unmodified E. coli tRNA(Lys) with affinity in the low micromolar range. MnmA does not bind observably to the nonsubstrate E. coli tRNA(Phe). Corroborating this, tRNA(Glu) protected MnmA from tryptic digestion. ATP also protected MnmA from trypsinolysis, suggesting the presence of an ATP binding site that is consistent with analysis of the amino acid sequence. We have reconstituted the in vitro biosynthesis of s(2)U using unmodified E. coli tRNA(Glu) as a substrate. The activity requires MnmA, Mg-ATP, l-cysteine, and the cysteine desulfurase IscS. HPLC analysis of thiolated tRNA digests using [(35)S]cysteine confirms that the product of the in vitro reaction is s(2)U. As in the case of 4-thiouridine synthesis, purified IscS-persulfide is able to provide sulfur for in vitro s(2)U synthesis in the absence of cysteine. Small RNAs that represent the anticodon stem loops for tRNA(Glu) and tRNA(Lys) are substrates of comparable activity to the full length tRNAs, indicating that the major determinants for substrate recognition are contained within this region.

Aminopeptidases↗

Magnesium-dependent alternative foldings of active and inactive Escherichia coli tRNA(Glu) revealed by chemical probing.

A stable conformer of Escherichia coli tRNA(Glu), obtained in the absence of Mg(2+), is inactive in the aminoacylation reaction. Probing it with diethylpyrocarbonate, dimethyl sulfate and ribonuclease V1 revealed that it has a hairpin structure with two internal loops; the helical segments at both extremities have the same structure as the acceptor stem and the anticodon arm of the native conformer of tRNA(Glu)and the middle helix is formed of nucleotides from the D-loop (G15-C20:2) and parts of the T-loop and stem (G51-C56), with G19 bulging out. This model is consistent with other known properties of this inactive conformer, including its capacity to dimerize. Therefore, this tRNA requires magnesium to acquire a conformation that can be aminoacylated, as others require a post-transcriptional modification to reach this active conformation.

Adenosine↗

The identification of spermine binding sites in 16S rRNA allows interpretation of the spermine effect on ribosomal 30S subunit functions.

A photoreactive analogue of spermine, N1-azidobenzamidino (ABA)-spermine, was covalently attached after irradiation to Escherichia coli 30S ribosomal subunits or naked 16S rRNA. By means of RNase H digestion and primer extension, the cross-linking sites of ABA-spermine in naked 16S rRNA were characterised and compared with those identified in 30S subunits. The 5' domain, the internal and terminal loops of helix H24, as well as the upper part of helix H44 in naked 16S rRNA, were found to be preferable binding sites for polyamines. Association of 16S rRNA with ribosomal proteins facilitated its interaction with photoprobe, except for 530 stem-loop nt, whose modification by ABA-spermine was abolished. Association of 30S with 50S subunits, poly(U) and AcPhe-tRNA (complex C) further altered the susceptibility of ABA-spermine cross-linking to 16S rRNA. Complex C, modified in its 30S subunit by ABA-spermine, reacted with puromycin similarly to non-photolabelled complex. On the contrary, poly(U)-programmed 70S ribosomes reconstituted from photolabelled 30S subunits and untreated 50S subunits bound AcPhe-tRNA more efficiently than untreated ribosomes, but were less able to recognise and reject near cognate aminoacyl-tRNA. The above can be interpreted in terms of conformational changes in 16S rRNA, induced by the incorporation of ABA-spermine.

Azides↗

Ribosomal proteins S12 and S13 function as control elements for translocation of the mRNA:tRNA complex.

Translocation of the mRNA:tRNA complex through the ribosome is promoted by elongation factor G (EF-G) during the translation cycle. Previous studies established that modification of ribosomal proteins with thiol-specific reagents promotes this event in the absence of EF-G. Here we identify two small subunit interface proteins S12 and S13 that are essential for maintenance of a pretranslocation state. Omission of these proteins using in vitro reconstitution procedures yields ribosomal particles that translate in the absence of enzymatic factors. Conversely, replacement of cysteine residues in these two proteins yields ribosomal particles that are refractive to stimulation with thiol-modifying reagents. These data support a model where S12 and S13 function as control elements for the more ancient rRNA- and tRNA-driven movements of translocation.

Biological Transport↗

Regulation of nuclear receptor activity by a pseudouridine synthase through posttranscriptional modification of steroid receptor RNA activator.

Nuclear receptors (NRs) induce transcription through association with coactivator complexes. We identified a pseudouridine synthase (PUS), mPus1p, as a coactivator for retinoic acid receptor (mRAR)gamma and other NR-dependent transactivation. mPus1p is a member of the truA subfamily of PUSs, a class of enzymes that isomerize uridine to pseudouridine in noncoding RNAs, such as tRNA, to ensure proper folding and function. mPus1p binds the first zinc finger of mRARgamma and also associates with other NRs. Interestingly, mPus1p pseudouridylates coactivator Steroid Receptor RNA Activator (SRA), and when coexpressed, mPus1p and SRA cooperatively enhance mRARgamma-mediated transcription. mPus1p, mRARgamma, and SRA exist in a retinoid-independent, promoter bound complex in the nucleus although mPus1p is also expressed in the nucleolus, where it likely modifies tRNA. Finally, we show that mPus1p-coactivator function required SRA, mPus1p-associated mRARgamma binding, and PUS activities. mPus1p-dependent pseudouridylation of SRA represents an additional type of posttranscriptional modification of a NR-coactivator complex that is important for NR signaling.

Animals↗

Hijacking pre-tRNA enables LTR-retrotransposon-initiated constitutive heterochromatin formation.

Pericentric heterochromatin serves as a fundamental component of eukaryotic chromosomes, endowing specialized genomic architecture with broad functional consequences. Although it is universally marked by H3K9me3 modification, the underlying pericentric DNA sequences diverge substantially across species. Here, by leveraging a transposition reporter system combined with a genome-wide RNA interference (RNAi) screen, we identified a specialized mechanism for recruiting SUV39H methyltransferase to initiate pericentric heterochromatin formation. This pathway depends on a highly ordered complex comprising the Puf68, pre-transfer RNAs (tRNAs), and the primer binding site (PBS). Puf68 binds with high affinity to poly-U tracts in pre-tRNA 3' trailer, forming a Puf68/pre-tRNA complex that subsequently base-pairs with the PBS of nascent long terminal repeat (LTR)-retrotransposons. Through direct interaction, Puf68 recruits Su(var)3-9 to these regions, catalyzing H3K9 trimethylation. Notably, Puf68 is sufficient to initiate de novo heterochromatin assembly both at pericentric and ectopically integrated LTR-retrotransposon regions. Our findings not only uncover a previously unrecognized mechanism of heterochromatin initiation but also resolve a long-standing question of how hosts harness nascent LTR-retrotransposon transcripts.

Heterochromatin↗

Three-dimensional models of the tRNA-like 3' termini of some plant viral RNAs.

Various plant viral RNAs possess a 3' terminus with tRNA-like properties. These viral RNAs are charged with an amino acid upon incubation with the cognate aminoacyl-tRNA synthetase and ATP. We have studied the structure of end-labelled 3'-terminal fragments of turnip yellow mosaic virus RNA and brome mosaic virus RNA 2 with chemical modifications of the adenosine and cytidine residues and with enzymatic digestions using RNase T1, nuclease S1 and the double-strand-specific ribonuclease from cobra venom. The data indicate that the 3' termini of these plant viral RNAs lack a cloverleaf structure as found in classical tRNA. The three-dimensional folding, however, reveals a striking resemblance with classical tRNA. The models proposed are supported by phylogenetic data. Apparently distinct three-dimensional solutions have evolved to meet the requirements for faithful recognition by tRNA-specific enzymes. The way in which the aminoacyl acceptor arms of these tRNA-like structures are constructed reveal novel features in RNA folding which may have a bearing on the secondary and tertiary structures of RNA in general. The dynamic behaviour of brome mosaic virus RNA 2 in solution presumably is illustrative of conformational transitions, which RNAs generally undergo on changing the ionic conditions.

Models, Molecular↗

Enzymatic formation of N2,N2-dimethylguanosine in eukaryotic tRNA: importance of the tRNA architecture.

In eukaryotic tRNA, guanosine at position 26 in the junction between the D-stem and the anticodon stem is mostly modified to N2,N2-dimethylguanosine (m2(2)G26). Here we review the available information on the enzyme catalyzing the formation of this modified nucleoside, the SAM-dependent tRNA (m2(2)G26)-methyltransferase, and our attemps to identify the parameters in tRNA needed for efficient enzymatic dimethylation of guanosine-26. The required identity elements in yeast tRNA for dimethylation under in vitro conditions by the yeast tRNA(m2(2)G26)-methyltransferase (the TRM1 gene product) are comprised of two G-C base pairs at positions G10-C25 and C11-G24 in the D-stem together with a variable loop of at least five nucleotides. These positive determinants do not seem to act via base specific interactions with the methyltransferase; they instead ensure that G26 is presented to the enzyme in a favorable orientation, within the central 3D-core of the tRNA molecule. The anticodon stem and loop is not involved in such an interaction with the enzyme. In a heterologous in vivo system, consisting of yeast tRNAs microinjected into Xenopus laevis oocytes, the requirements for modification of G26 are less stringent than in the yeast homologous in vitro system. Indeed, G26 in several microinjected tRNAs becomes monomethylated, while in yeast extracts it stays unmethylated, even after extensive incubation. Thus either the X laevis tRNA(m2(2)G26)-methyltransferase has a more relaxed specificity than its yeast homolog, or there exist two distinct G26-methylating activities, one for G26-monomethylation, and one for dimethylation of G26.(ABSTRACT TRUNCATED AT 250 WORDS)

Guanosine↗

The Kluyveromyces lactis gamma-toxin targets tRNA anticodons.

Kluyveromyces lactis killer strains secrete a heterotrimeric toxin (zymocin), which causes an irreversible growth arrest of sensitive yeast cells. Despite many efforts, the target(s) of the cytotoxic gamma-subunit of zymocin has remained elusive. Here we show that three tRNA species tRNA(Glu)(mcm(5)s(2)UUC), tRNA(Lys)(mcm(5)s(2)UUU), and tRNA(Gln)(mcm(5)s(2)UUG) are the targets of gamma-toxin. The toxin inhibits growth by cleaving these tRNAs at the 3' side of the modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). Transfer RNA lacking a part of or the entire mcm(5) group is inefficiently cleaved by gamma-toxin, explaining the gamma-toxin resistance of the modification-deficient trm9, elp1-elp6, and kti11-kti13 mutants. The K. lactis gamma-toxin is the first eukaryotic toxin shown to target tRNA.

Anticodon↗

On the function of modified nucleosides in the RNA world.

Presumably ribosome and transfer RNA (tRNA) evolved from a pre-existing function in the RNA stage of life and were secondarily adapted for protein synthesis. Various possible initial functions of the primitive ribosome (protoribosome) have been suggested. The initial function of the primitive ribosome and primitive genetic translation would have been quite similar. It is possible that, initially, both functions coexisted in the protoribosome. Given that the three-dimensional structure of ribosomal RNAs shows only minor variations throughout time, it is, then, most likely that present ribosomes can still recall (remember) the most important parts of the mechanism of their initial function. A process would have arisen to inactivate the initial function of the protoribosome without affecting genetic translation: the modification of some ribosome nucleosides. We suggest that the modifications of some rRNA nucleosides located in the catalytic center responsible for the initial function of primitive ribosomes, and of some of the tRNA nucleosides which interacted with the same center could have resulted in the inability of their recognition and secondary interaction. Thus, it is a known fact that the establishing of hydrogen bonds between modified nucleosides is rare and unstable. Therefore, the initial biological function of primitive ribosomes could have been inactivated without significantly affecting its three-dimensional structure. Therefore, without affecting the primitive translation. After the emergence of translation, some catalytic proteins (enzymes) which could modify the nucleosides of ribozymes could have arisen. In brief, we suggest that the catalytic proteins, through nucleoside modification, inactivated the catalytic RNA activity but RNA capacity to recognize and to bind other RNAs was not essentially altered. Only a few ribozymes were slightly affected by the modifications and they still maintain catalytic and binding activities. Therefore, we suggest that the proteins, through modification process, could have diminished the diverse functional capacities of the first RNA molecules. Auto-organization of the organic matter could be based on this type of interaction between macromolecules (protein and RNA).

Animals↗

Mitochondrial DNA of Hydra attenuata (Cnidaria): a sequence that includes an end of one linear molecule and the genes for l-rRNA, tRNA(f-Met), tRNA(Trp), COII, and ATPase8.

The 3231-nucleotide-pair (ntp) sequence of one end of one of the two linear mitochondrial (mt) DNA molecules of Hydra attenuata (phylum Cnidaria, class Hydrozoa, order Anthomedusae) has been determined. This segment contains complete genes for tRNA(f-Met), l-rRNA, tRNA(Trp), subunit 2 of cytochrome c oxidase (COII), subunit 8 of ATP synthetase (ATPase8), and the 5' 136 ntp of ATPase6. These genes are arranged in the order given and are transcribed from the same strand of the molecule. As in two other cnidarians, the hexacorallian anthozoan Metridium senile and the octocorallian anthozoan Sarcophyton glaucum, the mt-genetic code of H. attenuata is near standard. The only modification appears to be that TGA specifies tryptophan rather than termination. Also as in M. senile and S. glaucum, the encoded H. attenuata mt-tRNA(f-Met) has primary and secondary structural features resembling those of Escherichia coli initiator tRNA(t-Met). As the encoded mt-tRNA(Trp) cannot be folded into a totally orthodox secondary structure, two alternative forms are suggested. The encoded H. attenuata mt-l-rRNA is 1738 nt, which is 451 nt shorter than the M. senile mt-l-rRNA. Comparisons of secondary structure models of these two mt-l-rRNAs indicate that most of the size difference results from loss of nucleotides in the H. attenuata molecule at a minimum of 46 locations, which includes elimination of six distinct helical elements.

Adenosine Triphosphatases↗

A heterologous system for detecting eukaryotic enzymes which synthesize pseudouridine in transfer ribonucleic acids.

tRNA pseudouridylation activities have been detected in embryonic mouse cell fractions and in extracts from HeLa, mouse L-cell and baby hamster kidney (BHK) cell lines. These activities were identified by the use of heterologous reaction systems, with tRNA from hisT strains of Salmonella typhimurium as substrate. hisT mutants are defective for an enzyme that forms psi residues in the anticodon region of many tRNAs and accumulate undermodified species of tRNA. The pseudouridylation activity from BHK cells has been examined in detail and quantitated by a modified tritium release assay (Cortese, R., Kammen, H.O., Spengler, S.J., and Ames, B.N. (1974) J. Biol. Chem. 249, 1103-1108). Maximal rates of tritium release required a suitable cationic environment (optimally, a combination of Mg2+ and NH4+) and a thiol reductant. The activity was totally inhibited in the presence of thiol-reactive reagents, such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuribenzoate. A major portion of this 3H release activity was associated with psi modification reactions. This conclusion stems from the following observations: (a) BHK extracts preferentially catalyzed a release of 3H from hisT [5-3H]tRNA, rather than from similarly labeled wild type tRNA; (b) this activity was specific for protons attached to C5 of the pyrimidine rings; no release of 3H was obtained with hisT or wild type [6-3H]tRNA as substrate; (c) the reaction products of hisT tRNA with BHK enzyme were examined by reverse phase column chromatography of tRNAPhe isoacceptors on RPC-5 columns. The enzyme modified both of the principal isoacceptors of hisT tRNAPhe to an equal extent, yielding products indistinguishable from wild type tRNAPhe. Significant levels of 3H release were obtained by the action of enzyme on wild type [5-3H]tRNA, even after gel filtration of the enzyme. This suggests that the enzyme may be able to hypermodify certain species of wild type S. typhimurium tRNA. The activities for wild type tRNA and hisT tRNA appeared to be associated with the same enzyme.

Base Sequence↗

[Conformation mobility of the universal acceptor end of tRNA].

The conformation analysis of tRNA acceptor fragment XCCA (X=A) was performed. The ribose conformation was taken rigid C(3')-endo. The optimum conformation was found for the ACCA fragment. It was shown that the omega, omega'-angle of the terminal adenine in any crystal modification of the yeast tRNAFen is not optimal. The conformation mobility of the 3'-end ACCA fragment was calculated in the vicinity of optimum conformation. Redundant energy required for keeping the correct 3'-terminal tRNA orientation about the enzyme catalytic centre during any small orientation distortions of the non-universal X-nucleotide relative to the enzyme was estimated. The distortions may occur when non-gomologous tRNA interact with aminoacyl-tRNA-synthetase. It was found that redundant energy for 3' displacement is very anisotropic and some small X-nucleotide reorientation may decrease the catalitic velocity of aminoacylation u0(4)--10(5) times.

Mathematics↗

Involvement of tRNA in retrovirus expression: biological implications of reverse transcriptase-primer tRNA interactions.

We have previously studied the topographical and functional implications of the recognition of primer tRNATrp by avian retrovirus reverse transcriptase. Here we have presented evidence that the enzyme is able to deacylate beef liver Trp-tRNATrp, provided that 35-S viral RNA is present in the incubation mixture. No effect of dNTPs on this activity was observed. The extensive modification of tRNATrp with acrylonitrile led to a marked loss of priming activity by tRNATrp if the annealing between primer and template was performed at 37 degrees C, while the annealing of cyanoethylated tRNA with the viral genome at 75 degrees C gave almost normal levels of cDNA synthesis. We have also studied the priming behaviour of tRNATrp, modified by incorporation of various analogs of adenosine. Only tRNATrp-2'dA was active in cDNA initiation; 3'dA, 3'NH2-3'dA, and primer tRNA with formycin in the 3' end showed low or nonexistent priming activity.

Adenosine Triphosphate↗