Inaccuracy and the recognition of tRNA.
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Amino Acyl-tRNA Synthetases↗
Biomedical subjects
D Söll
Publications and source records attributed to D Söll.
Expression of the Synechocystis sp. strain PCC 6803 tRNA(Glu) gene provides tRNA for protein and chlorophyll biosynthesis.
In the cyanobacterium Synechocystis sp. strain PCC 6803 (Synechocystis 6803) delta-aminolevulinic acid (ALA), the sole precursor for the synthesis of the porphyrin rings of heme and chlorophyll, is formed from glutamate activated by acylation to tRNA(Glu) (G. P. O'Neill, D. M. Peterson, A. Schön, M. W. Chen, and D. Söll, J. Bacteriol. 170:3810-3816, 1988; S. Rieble and S. I. Beale, J. Biol. Chem. 263:8864-8871, 1988). We report here that Synechocystis 6803 possesses a single tRNA(Glu) gene which was transcribed as monomeric precursor tRNA and matured into the two tRNA(Glu) species. They differed in the extent of modification of the first anticodon base, 5-methylaminomethyl-2-thiouridine (O'Neill et al., 1988). The two tRNA species had equivalent capacities to stimulate the tRNA-dependent formation of ALA in Synechocystis 6803 and to provide glutamate for protein biosynthesis in an Escherichia coli-derived translation system. These results are in support of a dual role of tRNA(Glu). The levels of tRNA(Glu) were examined by Northern (RNA) blot analysis of cellular RNA and by aminoacylation assays in cultures of Synechocystis 6803 in which the amount of chlorophyll synthesized was modulated over a 10-fold range by various illumination regimens or by the addition of inhibitors of chlorophyll and ALA biosynthesis. In these cultures, the level of tRNA(Glu) was always a constant fraction of the total tRNA population, suggesting that tRNA(Glu) and chlorophyll levels are regulated independently. In addition, the tRNA(Glu) was always fully aminoacylated in vivo.
Aminolevulinic Acid↗
Transfer RNA and the formation of the heme and chlorophyll precursor, 5-aminolevulinic acid.
5-Aminolevulinic acid is the first committed precursor for the synthesis of porphyrins such as hemes and chlorophylls. In many organisms aminolevulinate is synthesized from glutamate in a three-step pathway (C5 pathway). The key step in this conversion is a tRNA-mediated reduction of glutamate to glutamate-1-semialdehyde. tRNA is a specific cofactor for an NADPH-dependent enzyme, Glu-tRNA reductase, which is capable of sequence-specific recognition of Glu-tRNA(Glu). tRNA(Glu) is a dual-function molecule; it participates both in protein and in aminolevulinate biosynthesis. This reduction reaction represents a novel role for tRNA where it participates in a metabolic conversion of its amino acid into a low molecular weight metabolite which is subsequently not used in peptide bond synthesis.
Aldehyde Oxidoreductases↗
A selection for mutants of the RNA polymerase III transcription apparatus: PCF1 stimulates transcription of tRNA and 5S RNA genes.
A genetic approach has been developed to study transcription by RNA polymerase III. A pair of Schizosaccharomyces pombe nonsense suppressor tRNA genes were arranged in tandem such that expression of the downstream (supS1) tRNA suppressor was dependent upon transcription initiated by the internal promoter of the upstream (sup9-e) gene. Dominant mutant strains of Saccharomyces cerevisiae were isolated that suppress in trans the effect of an A block promoter mutation (A19) in the sup9-e gene and restore supS1 suppressor activity. Fifteen mutant strains, eight of which were independently isolated, all have elevated steady-state levels of sup9-e A19 RNA consistent with an increase in gene transcription. Extracts of a strain carrying the dominant mutant gene, PCF1, show a general 6-fold stimulation in transcription of mutant (A19) and wild-type tRNA genes and increase 5S gene transcription 4-fold compared with extracts from a wild-type strain. A transcription factor exclusion assay was used to show that the PCF1 mutation affects two distinct stages in transcription: one prior to and one after stable complex formation; and that these effects are mediated by a component of the stable complex. Further evidence of an effect during complex assembly was obtained in a time-course experiment that showed a shortened lag phase in the PCF1 extract. The results indicate that PCF1 is either a component of the stable complex or a positive regulator of its activity.
Base Sequence↗
Structure of E. coli glutaminyl-tRNA synthetase complexed with tRNA(Gln) and ATP at 2.8 A resolution.
The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) complexed with its cognate glutaminyl transfer RNA (tRNA(Gln] and adenosine triphosphate (ATP) has been derived from a 2.8 angstrom resolution electron density map and the known protein and tRNA sequences. The 63.4-kilodalton monomeric enzyme consists of four domains arranged to give an elongated molecule with an axial ratio greater than 3 to 1. Its interactions with the tRNA extend from the anticodon to the acceptor stem along the entire inside of the L of the tRNA. The complexed tRNA retains the overall conformation of the yeast phenylalanine tRNA (tRNA(Phe] with two major differences: the 3' acceptor strand of tRNA(Gln) makes a hairpin turn toward the inside of the L, with the disruption of the final base pair of the acceptor stem, and the anticodon loop adopts a conformation not seen in any of the previously determined tRNA structures. Specific recognition elements identified so far include (i) enzyme contacts with the 2-amino groups of guanine via the tRNA minor groove in the acceptor stem at G2 and G3; (ii) interactions between the enzyme and the anticodon nucleotides; and (iii) the ability of the nucleotides G73 and U1.A72 of the cognate tRNA to assume a conformation stabilized by the protein at a lower free energy cost than noncognate sequences. The central domain of this synthetase binds ATP, glutamine, and the acceptor end of the tRNA as well as making specific interactions with the acceptor stem.2+t is
Adenosine Triphosphate↗
Structural basis for misaminoacylation by mutant E. coli glutaminyl-tRNA synthetase enzymes.
A single-site mutant of Escherichia coli glutaminyl-synthetase (D235N, GlnRS7) that incorrectly acylates in vivo the amber suppressor supF tyrosine transfer RNA (tRNA(Tyr] with glutamine has been described. Two additional mutant forms of the enzyme showing this misacylation property have now been isolated in vivo (D235G, GlnRS10; I129T, GlnRS15). All three mischarging mutant enzymes still retain a certain degree of tRNA specificity; in vivo they acylate supE glutaminyl tRNA (tRNA(Gln] and supF tRNA(Tyr) but not a number of other suppressor tRNA's. These genetic experiments define two positions in GlnRS where amino acid substitution results in a relaxed specificity of tRNA discrimination. The crystal structure of the GlnRS:tRNA(Gln) complex provides a structural basis for interpreting these data. In the wild-type enzyme Asp235 makes sequence-specific hydrogen bonds through its side chain carboxylate group with base pair G3.C70 in the minor groove of the acceptor stem of the tRNA. This observation implicates base pair 3.70 as one of the identity determinants of tRNA(Gln). Isoleucine 129 is positioned adjacent to the phosphate of nucleotide C74, which forms part of a hairpin structure adopted by the acceptor end of the complexed tRNA molecule. These results identify specific areas in the structure of the complex that are critical to accurate tRNA discrimination by GlnRS.
Acylation↗
Multiple mutations of the first gene of a dimeric tRNA gene abolish in vitro tRNA gene transcription.
Eukaryotic tRNA expression initiates with transcription by RNA polymerase III and requires two additional protein factors and two regions within the tRNA gene (the 5'-internal control region (ICR) or A-box and the 3'-ICR or B-box). Using a reconstituted Saccharomyces cerevisiae RNA polymerase III system, the transcription of various 5'-ICR, 3'-ICR, and double mutation alleles of the Schizosaccharomyces pombe sup3-e dimeric tRNA gene were studied. The sup3-e tRNA locus consists of an upstream serine tRNA gene and a downstream initiator methionine tRNA gene which are transcribed as a dimeric precursor and processed to give two tRNAs. Only the ICRs of the tRNA(Ser) gene are active in directing dimeric gene transcription. Mutations in the 3'-ICR of the tRNA(Ser) gene reduce transcription of the dimer more than those in the 5'-ICR. Mutations in the 5'-ICR were found which greatly increased or decreased transcription of the dimer, while base changes in the 3'-ICR were only found to decrease transcription. This suggests a modulatory role for the 5'-ICR in transcription regulation. Mutation of the methionine tRNA gene ICR has little effect on sup3-e transcription, and no detectable transcripts initiate from the methionine tRNA gene when the tRNA(Ser) gene promoter is inactivated by mutation. Comparison with transcription studies of other mutant tRNA genes suggests that nucleotides sites within the ICRs, such as nucleotides 8, 10, 13, 18, and 19 in the 5'-ICR and 48, 53, 56, 57, and 58 in the 3'-ICR, appear to have evolved universal importance for RNA polymerase III transcription in eukaryotes. Thus these ICR sequences may play a critical role in regulation of tRNA expression.
Cloning, Molecular↗
The selenocysteine-inserting opal suppressor serine tRNA from E. coli is highly unusual in structure and modification.
Selenocysteine is cotranslationally incorporated into selenoproteins in a unique pathway involving tRNA mediated suppression of a UGA nonsense codon (1-3). The DNA sequence of the gene for this suppressor tRNA from Escherichia coli predicts unusual features of the gene product (4). We determined the sequence of this serine tRNA (tRNA(UCASer]. It is the longest tRNA (95 nt) known to date with an acceptor stem of 8 base pairs and lacks some of the 'invariant' nucleotides found in other tRNAs. It is the first E. coli tRNA that contains the hypermodified nucleotide i6A, adjacent to the UGA-recognizing anticodon UCA. The implications of the unusual structure and modification of this tRNA on recognition by seryl-tRNA synthetase, by tRNA modifying enzymes, and on codon recognition are discussed.
Amino Acyl-tRNA Synthetases↗
Substrate structural requirements of Schizosaccharomyces pombe RNase P.
RNase P from Schizosaccharomyces pombe has been purified over 2000-fold. The apparent Km for two S. pombe tRNA precursors derived from the supS1 and sup3-e tRNA(Ser) genes is 20 nM; the apparent Vmax is 2.5 nM/min (supS1) and 1.1 nM/min (sup3-e). Processing studies with precursors of other mutants show that the structures of the acceptor stem and anticodon/intron loop of tRNA are crucial for S. pombe RNase P action.
Base Sequence↗
Characterization of cis-acting mutations which increase expression of a glnS-lacZ fusion in Escherichia coli.
glnS-lacZ fusions have been used to isolate mutations which enhance expression of the glnS gene. One mutation, acting at the level of transcription changes the -10 region of the promoter from GATCAT to TATCAT and produces a ten-fold increase in mRNA. Four other mutations which enhance expression three-fold to nine-fold fall within the transcribed region, but not within the Shine and Dalgarno sequence nor in the initiator codon. These mutations are shown to enhance translation specifically and different models are considered to explain their mode of action.
Amino Acid Sequence↗
delta-Aminolevulinic acid biosynthesis in Escherichia coli and Bacillus subtilis involves formation of glutamyl-tRNA.
Cell-free extracts of Escherichia coli and Bacillus subtilis catalyzed the tRNA-dependent, RNase A-sensitive formation of delta-aminolevulinic acid (ALA) from glutamate. Cell extracts prepared from cultures of E. coli grown under aerobic or anaerobic conditions had similar levels of ALA biosynthetic activity. Both the tRNA-stimulated conversion of glutamate to ALA and the conversion of glutamate-1-semialdehyde to ALA were inhibited by gabaculin. However, gabaculin had no effect on the growth of either E. coli or B. subtilis. The tRNA-dependent transformation of glutamate to ALA in E. coli and B. subtilis thus appears to be very similar to the pathway found in cyanobacteria, certain obligate anaerobic eubacteria, archaebacteria and in the chloroplasts of algae and higher plant species.
Alanine↗
The unusually long amino acid acceptor stem of Escherichia coli selenocysteine tRNA results from abnormal cleavage by RNase P.
The nucleotide sequence of the gene encoding the Escherichia coli selenocysteine tRNA (tRNA(SeCys] predicts an unusually long acceptor stem of 8 base pairs (one more than other tRNAs). Here we show by in vivo experiments (Northern blots, primer extension analysis) and by in vitro RNA processing studies that E. coli tRNA(SeCys) does contain this additional basepair, and that its formation results from abnormal cleavage by RNase P.
Base Composition↗
Accuracy of in vivo aminoacylation requires proper balance of tRNA and aminoacyl-tRNA synthetase.
The fidelity of protein biosynthesis in any cell rests on the accuracy of aminoacylation of tRNA. The exquisite specificity of this reaction is critically dependent on the correct recognition of tRNA by aminoacyl-tRNA synthetases. It is shown here that the relative concentrations of a tRNA and its cognate aminoacyl-tRNA synthetase are normally well balanced and crucial for maintenance of accurate aminoacylation. When Escherichia coli Gln-tRNA synthetase is overproduced in vivo, it incorrectly acylates the supF amber suppressor tRNA(Tyr) with Gln. This effect is abolished when the intracellular concentration of the cognate tRNA(Gln2) is also elevate. These data indicate that the presence of aminoacyl-tRNA synthetase and the cognate tRNAs in complexed form, which requires the proper balance of the two macromolecules, is critical in maintaining the fidelity of protein biosynthesis. Thus, limits exist on the relative levels of tRNAs and aminoacyl-tRNA synthetases within a cell.
Amino Acyl-tRNA Synthetases↗
The 5'-terminal guanylate of chloroplast histidine tRNA is encoded in its gene.
Among tRNA species histidine tRNAs possess the unique feature of having an additional nucleotide, a guanylate, at their 5'-end. In prokaryotes this G is encoded in the gene sequence and retained in the mature tRNA as result of an unusual cleavage by RNase P (Orellana, O., Cooley, L., and Söll, D. (1986) Mol. Cell. Biol. 6, 525-529), while in eukaryotes it is added post-transcriptionally by a special tRNA guanylyl transferase (Cooley, L., Appel, B., and Söll, D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6475-6479). Here we show that the additional G of chloroplast tRNAHis species is gene-encoded and retained in the tRNA by a processing step analogous to the prokaryotic one. However, the structural requirements for recognition by the different RNase P activities are not the same.
Animals↗
Overproduction and purification of Escherichia coli tRNA(2Gln) and its use in crystallization of the glutaminyl-tRNA synthetase-tRNA(Gln) complex.
We describe the genetically engineered overproduction of Escherichia coli tRNA(2Gln), its purification by high pressure liquid chromatography (HPLC), and its subsequent use in the growth of crystals of the E. coli glutaminyl-tRNA synthetase-tRNA(Gln) complex. The overproduced tRNA represents 60 to 70% of the total tRNA extracted from the engineered strain. A single anion exchange HPLC column is then sufficient to increase the purity of this isoacceptor to 90 to 95%. Crystals of this material complexed with the monomeric E. coli glutaminyl-tRNA synthetase enzyme were obtained by vapor diffusion from solutions containing sodium citrate as the precipitating agent. The crystals diffract to beyond 2.8 A resolution (1 A = 0.1 nm) and are of the orthorhombic space group C222(1) with unit cell parameters a = 240.5 A, b = 93.9 A, c = 115.7 A. Gel electrophoresis of dissolved crystals demonstrates the presence of both protein and tRNA.
Amino Acyl-tRNA Synthetases↗
Genetic code: enter a new amino acid.
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Base Sequence↗
Processing of histidine transfer RNA precursors. Abnormal cleavage site for RNase P.
The 5'-terminal guanylate residue (G-1) of mature Escherichia coli tRNA(His) is generated as a result of an unusual cleavage by RNase P (Orellana, O., Cooley, L., and Söll, D. (1986) Mol. Cell. Biol. 6, 525-529). We have examined the importance of the unique acceptor stem structure of E. coli tRNA(His) in determining the specificity of RNase P cleavage. Mutant tRNA(His) precursors bearing substitutions of the normal base G-1 or the opposing, potentially paired base, C73, can be cleaved at the +1 position, in contrast to wild-type precursors which are cut exclusively at the -1 position. These data indicate that the nature of the base at position -1 is of greater importance in determining the site of RNase P cleavage than potential base pairing between nucleotides -1 and 73. In addition, processing of the mutant precursors by M1-RNA or P RNA under conditions of ribozyme catalysis yields a higher proportion of +1-cleaved products in comparison to the reaction catalyzed by the RNase P holoenzyme. This lower sensitivity of the holoenzyme to alterations in acceptor stem structure suggests that the protein moiety of RNase P may play a role in determining the specificity of the reaction and implies that recognition of the substrate involves additional regions of the tRNA. We have also shown that the RNase P holoenzyme and tRNA(His) precursor of Saccharomyces cerevisiae, unlike their prokaryotic counterparts, do not possess these abilities to carry out this unusual reaction.
Endoribonucleases↗