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Ribosomal proteins are indispensable components of a living cell, and yet their structures are remarkably diverse in different species. Here we use manually curated structural alignments to provide a comprehensive catalog of structural variations in homologous ribosomal proteins from bacteria, archaea, eukaryotes, and eukaryotic organelles. By resolving numerous ambiguities and errors of automated structural and sequence alignments, we uncover a whole new class of structural variations that reside within seemingly conserved segments of ribosomal proteins. We then illustrate that these variations reflect an apparent adaptation of ribosomal proteins to the specific environments and lifestyles of living species. Finally, we show that most of these structural variations reside within nonglobular extensions of ribosomal proteins—protein segments that are thought to promote ribosome biogenesis by stabilizing the proper folding of ribosomal RNA. We show that although the extensions are thought to be the most ancient peptides on our planet, they are in fact the most rapidly evolving and most structurally and functionally diverse segments of ribosomal proteins. Overall, our work illustrates that, despite being long considered as slowly evolving and highly conserved, ribosomal proteins are more complex and more specialized than is generally recognized.

Cisplatin is a widely prescribed anticancer drug, which triggers cell death by covalent binding to a broad range of biological molecules. Among cisplatin targets, cellular RNAs remain the most poorly characterized molecules. Although cisplatin was shown to inactivate essential RNAs, including ribosomal, spliceosomal and telomeric RNAs, cisplatin binding sites in most RNA molecules are unknown, and therefore it remains challenging to study how modifications of RNA by cisplatin contributes to its toxicity. Here we report a 2.6Å-resolution X-ray structure of cisplatin-modified 70S ribosome, which describes cisplatin binding to the ribosome and provides the first nearly atomic model of cisplatin–RNA complex. We observe nine cisplatin molecules bound to the ribosome and reveal consensus structural features of the cisplatin-binding sites. Two of the cisplatin molecules modify conserved functional centers of the ribosome—the mRNA-channel and the GTPase center. In the mRNA-channel, cisplatin intercalates between the ribosome and the messenger RNA, suggesting that the observed inhibition of protein synthesis by cisplatin is caused by impaired mRNA-translocation. Our structure provides an insight into RNA targeting and inhibition by cisplatin, which can help predict cisplatin-binding sites in other cellular RNAs and design studies to elucidate a link between RNA modifications by cisplatin and cisplatin toxicity.

Protein biosynthesis requires aminoacyl-transfer RNA (tRNA) synthetases to provide aminoacyl-tRNA substrates for the ribosome. Most bacteria and all archaea lack a glutaminyl-tRNA synthetase (GlnRS); instead, Gln-tRNA(Gln) is produced via an indirect pathway: a glutamyl-tRNA synthetase (GluRS) first attaches glutamate (Glu) to tRNA(Gln), and an amidotransferase converts Glu-tRNA(Gln) to Gln-tRNA(Gln). The human pathogen Helicobacter pylori encodes two GluRS enzymes, with GluRS2 specifically aminoacylating Glu onto tRNA(Gln). It was proposed that GluRS2 is evolving into a bacterial-type GlnRS. Herein, we have combined rational design and directed evolution approaches to test this hypothesis. We show that, in contrast to wild-type (WT) GlnRS2, an engineered enzyme variant (M110) with seven amino acid changes is able to rescue growth of the temperature-sensitive Escherichia coli glnS strain UT172 at its non-permissive temperature. In vitro kinetic analyses reveal that WT GluRS2 selectively acylates Glu over Gln, whereas M110 acylates Gln 4-fold more efficiently than Glu. In addition, M110 hydrolyzes adenosine triphosphate 2.5-fold faster in the presence of Glu than Gln, suggesting that an editing activity has evolved in this variant to discriminate against Glu. These data imply that GluRS2 is a few steps away from evolving into a GlnRS and provides a paradigm for studying aminoacyl-tRNA synthetase evolution using directed engineering approaches.

Histidine transfer RNA (tRNA) is unique among tRNA species as it carries an additional nucleotide at its 5' terminus. This unusual G(-1) residue is the major tRNA(His) identity element, and essential for recognition by the cognate histidyl-tRNA synthetase to allow efficient His-tRNA(His) formation. In many organisms G(-1) is added post-transcriptionally as part of the tRNA maturation process. tRNA(His) guanylyltransferase (Thg1) specifically adds the guanylyate residue by recognizing the tRNA(His) anticodon. Thg1 homologs from all three domains of life have been the subject of exciting research that gave rise to a detailed biochemical, structural and phylogenetic enzyme characterization. Thg1 homologs are phylogenetically classified into eukaryal- and archaeal-type enzymes differing characteristically in their cofactor requirements and specificity. Yeast Thg1 displays a unique but limited ability to add 2-3 G or C residues to mutant tRNA substrates, thus catalyzing a 3' → 5' RNA polymerization. Archaeal-type Thg1, which has been horizontally transferred to certain bacteria and few eukarya, displays a more relaxed substrate range and may play additional roles in tRNA editing and repair. The crystal structure of human Thg1 revealed a fascinating structural similarity to 5' → 3' polymerases, indicating that Thg1 derives from classical polymerases and evolved to assume its specific function in tRNA(His) processing.

Archaea make glutaminyl-tRNA (Gln-tRNA(Gln)) in a two-step process; a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) forms Glu-tRNA(Gln), while the heterodimeric amidotransferase GatDE converts this mischarged tRNA to Gln-tRNA(Gln). Many prokaryotes synthesize asparaginyl-tRNA (Asn-tRNA(Asn)) in a similar manner using a non-discriminating aspartyl-tRNA synthetase (ND-AspRS) and the heterotrimeric amidotransferase GatCAB. The transamidosome, a complex of tRNA synthetase, amidotransferase and tRNA, was first described for the latter system in Thermus thermophilus [Bailly, M., Blaise, M., Lorber, B., Becker, H.D. and Kern, D. (2007) The transamidosome: a dynamic ribonucleoprotein particle dedicated to prokaryotic tRNA-dependent asparagine biosynthesis. Mol. Cell, 28, 228-239.]. Here, we show a similar complex for Gln-tRNA(Gln) formation in Methanothermobacter thermautotrophicus that allows the mischarged Glu-tRNA(Gln) made by the tRNA synthetase to be channeled to the amidotransferase. The association of archaeal ND-GluRS with GatDE (K(D) = 100 ± 22 nM) sequesters the tRNA synthetase for Gln-tRNA(Gln) formation, with GatDE reducing the affinity of ND-GluRS for tRNA(Glu) by at least 13-fold. Unlike the T. thermophilus transamidosome, the archaeal complex does not require tRNA for its formation, is not stable through product (Gln-tRNA(Gln)) formation, and has no major effect on the kinetics of tRNA(Gln) glutamylation nor transamidation. The differences between the two transamidosomes may be a consequence of the fact that ND-GluRS is a class I aminoacyl-tRNA synthetase, while ND-AspRS belongs to the class II family.

The RNA splicing and processing endonuclease from Nanoarchaeum equitans (NEQ) belongs to the recently identified (alphabeta)(2) family of splicing endonucleases that require two different subunits for splicing activity. N. equitans splicing endonuclease comprises the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Here, we report the crystal structure of the functional NEQ enzyme at 2.1 A containing both subunits, as well as that of the NEQ261 subunit alone at 2.2 A. The functional enzyme resembles previously known alpha(2) and alpha(4) endonucleases but forms a heterotetramer: a dimer of two heterodimers of the catalytic subunit (NEQ205) and the structural subunit (NEQ261). Surprisingly, NEQ261 alone forms a homodimer, similar to the previously known homodimer of the catalytic subunit. The homodimers of isolated subunits are inhibitory to heterodimerization as illustrated by a covalently linked catalytic homodimer that had no RNA cleavage activity upon mixing with the structural subunit. Detailed structural comparison reveals a more favorable hetero- than homodimerization interface, thereby suggesting a possible regulation mechanism of enzyme assembly through available subunits. Finally, the uniquely flexible active site of the NEQ endonuclease provides a possible explanation for its broader substrate specificity.

Aminoacyl-tRNAs (aa-tRNAs) are the essential substrates for translation. Most aa-tRNAs are formed by direct aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases. However, a smaller number of aa-tRNAs (Asn-tRNA, Gln-tRNA, Cys-tRNA and Sec-tRNA) are made by synthesizing the amino acid on the tRNA by first attaching a non-cognate amino acid to the tRNA, which is then converted to the cognate one catalyzed by tRNA-dependent modifying enzymes. Asn-tRNA or Gln-tRNA formation in most prokaryotes requires amidation of Asp-tRNA or Glu-tRNA by amidotransferases that couple an amidase or an asparaginase to liberate ammonia with a tRNA-dependent kinase. Both archaeal and eukaryotic Sec-tRNA biosynthesis and Cys-tRNA synthesis in methanogens require O-phosophoseryl-tRNA formation. For tRNA-dependent Cys biosynthesis, O-phosphoseryl-tRNA synthetase directly attaches the amino acid to the tRNA which is then converted to Cys by Sep-tRNA: Cys-tRNA synthase. In Sec-tRNA synthesis, O-phosphoseryl-tRNA kinase phosphorylates Ser-tRNA to form the intermediate which is then modified to Sec-tRNA by Sep-tRNA:Sec-tRNA synthase. Complex formation between enzymes in the same pathway may protect the fidelity of protein synthesis. How these tRNA-dependent amino acid biosynthetic routes are integrated into overall metabolism may explain why they are still retained in so many organisms.

The micronutrient selenium is present in proteins as selenocysteine (Sec). In eukaryotes and archaea, Sec is formed in a tRNA-dependent conversion of O-phosphoserine (Sep) by O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS). Here, we present the crystal structure of Methanococcus maripaludis SepSecS complexed with PLP at 2.5 A resolution. SepSecS, a member of the Fold Type I PLP enzyme family, forms an (alpha2)2 homotetramer through its N-terminal extension. The active site lies on the dimer interface with each monomer contributing essential residues. In contrast to other Fold Type I PLP enzymes, Asn247 in SepSecS replaces the conserved Asp in binding the pyridinium nitrogen of PLP. A structural comparison with Escherichia coli selenocysteine lyase allowed construction of a model of Sep binding to the SepSecS catalytic site. Mutations of three conserved active site arginines (Arg72, Arg94, Arg307), protruding from the neighboring subunit, led to loss of in vivo and in vitro activity. The lack of active site cysteines demonstrates that a perselenide is not involved in SepSecS-catalyzed Sec formation; instead, the conserved arginines may facilitate the selenation reaction. Structural phylogeny shows that SepSecS evolved early in the history of PLP enzymes, and indicates that tRNA-dependent Sec formation is a primordial process.

5-Aminolevulinic acid (ALA) is a precursor in the biosynthesis of tetrapyrroles including chlorophylls and heme. The formation of ALA involves two enzymatic steps which take place in the chloroplast in plants. The first enzyme, glutamyl-tRNA reductase, and the second enzyme, glutamate-1-semialdehyde-2,1-aminomutase, are encoded by the nuclearHEMA and GSA genes, respectively. To assess the significance of the HEMA gene for chlorophyll and heme synthesis, transgenic Arabidopsis plants that expressed antisense HEMA1 mRNA from the constitutive cauliflower mosaic virus 35S promoter were generated. These plants exhibited varying degrees of chlorophyll deficiency, ranging from patchy yellow to total yellow. Analysis indicated that these plants had decreased levels of chlorophyll, non-covalently bound hemes, and ALA; their levels were proportional to the level of glutamyl-tRNA reductase expression and were inversely related to the levels of antisenseHEMA transcripts. Plants that lacked chlorophyll failed to survive under normal growth conditions, indicating thatHEMA gene expression is essential for growth.

Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs ensure both accurate RNA recognition and the efficient catalysis of aminoacylation. The effects of tRNA(Trp)variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthe-tase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken. This showed that tRNA(Trp)identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl-adenylate, but predominantly affects the rate at which trypto-phan is transferred from TrpRS-tryptophanyl adenylate to tRNA. Use of the binding ( K (tRNA)) and rate constants ( k (4)) to determine the energetic levels of the various species in the aminoacylation reaction showed a difference of approximately 2 kcal mol(-1)in the barrier to transition state formation compared to wild-type for both tRNA(Trp)A-->C73 and. These results directly show that tRNA identity contributes to the degree of complementarity to the transition state for tRNA charging in the active site of an aminoacyl-tRNA synthetase:aminoacyl-adenylate:tRNA complex.

In many organisms (e.g., gram-positive eubacteria) Gin-tRNA is not formed by direct glutaminylation of tRNAGln but by a specific transamidation of Glu-tRNAGln. We wondered whether a similar transamidation pathway also operates in the formation of Asn-tRNA in these organisms. Therefore we tested in S-100 preparations of Lactobacillus bulgaricus, a gram-positive eubacterium, for the conversion by an amidotransferase of [14C]Asp-tRNA to [14C]Asn-tRNA. As no transamidation was observed, we searched for genes for asparaginyl-tRNA synthetase (AsnRS). Two DNA fragments (from different locations of the L.bulgaricus chromosome) were found each containing an ORF whose sequence resembled that of the Escherichia coli asnS gene. The derived amino acid sequences of the two ORFs (432 amino acids) were the same and 41% identical with E.coli AsnRS. When one of the ORFs was expressed in E.coli, it complemented the temperature sensitivity of an E.coli asnS mutant. S-100 preparations of this transformant showed increased charging of unfractionated L.bulgaricus tRNA with asparagine. Deletion of the 3'-terminal region of the L.bulgaricus AsnRS gene led to loss of its complementation and aminoacylation properties. This indicates that L.bulgaricus contains a functional AsnRS. Thus, the transamidation pathway operates only for Gin-tRNAGln formation in this organism, and possibly in all gram-positive eubacteria.

Arabidopsis thaliana roots grow in a wavy pattern on inclined agar plates. This waving behaviour has been interpreted as representing a gravitropism-dependent thigmotropic response. We argue instead that this root waving represents primarily a flattened spiral growth pattern resulting from circumnutation and gravitropism

Aminoacyl-tRNA synthetases interact with their cognate tRNAs in a highly specific fashion. We have examined the phenomenon that upon complex formation E. coli glutaminyl-tRNA synthetase destabilizes tRNA(Gln) causing chain scissions in the presence of Mg2+ ions. The phosphodiester bond cleavage produces 3'-phosphate and 5'-hydroxyl ends. This kind of experiment is useful for detecting conformational changes in tRNA. Our results show that the cleavage is synthetase-specific, that mutant and wild-type tRNA(Gln) species can assume a different conformation, and that modified nucleosides in tRNA enhance the structural stability of the molecule.

Genetic encoding of noncanonical amino acids (ncAAs) into proteins is a powerful approach to study protein functions. Pyrrolysyl-tRNA synthetase (PylRS), a polyspecific aminoacyl-tRNA synthetase in wide use, has facilitated incorporation of a large number of different ncAAs into proteins to date. To make this process more efficient, we rationally evolved tRNA(Pyl) to create tRNA(Pyl-opt) with six nucleotide changes. This improved tRNA was tested as substrate for wild-type PylRS as well as three characterized PylRS variants (N(ϵ)-acetyllysyl-tRNA synthetase [AcKRS], 3-iodo-phenylalanyl-tRNA synthetase [IFRS], a broad specific PylRS variant [PylRS-AA]) to incorporate ncAAs at UAG codons in super-folder green fluorescence protein (sfGFP). tRNA(Pyl-opt) facilitated a 5-fold increase in AcK incorporation into two positions of sfGFP simultaneously. In addition, AcK incorporation into two target proteins (Escherichia coli malate dehydrogenase and human histone H3) caused homogenous acetylation at multiple lysine residues in high yield. Using tRNA(Pyl-opt) with PylRS and various PylRS variants facilitated efficient incorporation of six other ncAAs into sfGFP. Kinetic analyses revealed that the mutations in tRNA(Pyl-opt) had no significant effect on the catalytic efficiency and substrate binding of PylRS enzymes. Thus tRNA(Pyl-opt) should be an excellent replacement of wild-type tRNA(Pyl) for future ncAA incorporation by PylRS enzymes.

Sequencing of a large number of microbial genomes has led to the discovery of new enzymes involved in tRNA biosynthesis and tRNA function. Preparation of a great variety of RNA molecules is, therefore, of major interest for biochemical characterization of these proteins. We describe a fast, cost-effective and efficient method for in vitro production of tRNA transcripts. T7 RNA polymerase requires a double-stranded DNA promoter in order to initiate transcription; however, elongation does not require a double-stranded DNA template. A partially double-stranded transcription template formed by annealing of a short oligonucleotide, complementary to the T7 promoter, to a larger oligonucleotide is shown to be a good substrate for in vitro transcription. This method allows rapid production of a variety of tRNA transcripts which can be aminoacylated well. This eliminates the need for cloning of tRNA genes, large-scale plasmid preparation and enzymatic digestion.

Transcription of isolated repeat units of D. melanogaster 5S DNA in a Drosophila KcO cell extract revealed three types of template activities. 5SI DNA encodes the known 5S rRNA of D. melanogaster and has a relatively high transcription efficiency. 5SII DNA is identical to 5SI DNA except for a two-nucleotide deletion at 5S rRNA positions 28 and 29; the efficiency of transcription is approximately 40% that of 5SI DNA and because of the deletion, the primary transcript is two nucleotides shorter. 5SIII DNA does not support in vitro transcription (less than 2% 5SI DNA), but has the same sequence as 5SI DNA except for a single G to A transition at position 86. This is the first reported point-mutation in a 5S RNA gene resulting in loss of transcription function. Of approximately 23 5S rRNA gene copies in a cloned 5S DNA sub-cluster (p12D1) 19 appear to be of the transcriptionally inactive 5SIII DNA type.

Each eukaryotic cell contains about 100 different transfer RNA species, which are polynucleotides whose chainlength varies from 73 to 93 (Sprinzl et al. 1980). Transfer RNAs play a key role in protein biosynthesis: They carry the amino acids to the ribosome, where they pair with messenger RNA to ensure the correct placement of each amino acid into the growing polypeptide chain. The structural features of tRNA, therefore, must contain all the information for specific interactions with many components in the protein-synthesizing machinery. The number of specific interactions of tRNA is even greater when one considers the complex sequence of enzymatic steps involved in the biosynthesis of these molecules (Figure 1). The tRNA genes are transcribed into larger precursor RNA molecules, which are subsequently cleaved to mature-size tRNA by the action of special processing nucleases. Another part of this maturation process is the synthesis of modified nucleosides, which proceeds as posttranscriptional nucleotide modification. The study of tRNA biosynthesis is very difficult since the steady-state level of tRNA precursors in normal cells is low. The biochemical approach to this problem was made possible by recent methodological developments in recombinant DNA research and DNA sequence analysis. Insight into the structure of tRNA genes, coding for a variety of tRNAs, allowed the search for conserved sequences and attempts to define transcription regulatory signals in the DNA. The use of tRNA genes with known nucleotide sequence led to the characterization of enzymatic systems, which accurately transcribed such genes into tRNA precursors. These RNA species can then be used to unravel the

We describe a simple method for specific chemical labeling of DNA fragments at their 3'-termini. The procedure includes enzymatic addition of 4-thiouridine, followed by reaction in mild non-denaturing conditions with the highly reactive alpha-haloacetamido derivatives of several chemical labels. The attached reporter molecule can be removed by extended treatment with beta-mercaptoethanol. Among the potential applications of this labeling method is the study of specific protein-DNA interactions in solution.

Transcription of eukaryotic tRNA genes is controlled by two intragenic regions, the D-control region (which in the tRNA codes for the D-stem and -loop) and the T-control region (which in the tRNA codes for the T psi C loop). To determine whether these sequences alone are sufficient to promote tRNA gene transcription in vitro, the two control regions of a Drosophila tRNAArg gene were cloned separately from the context of the parental DNA (these constructions are called tRNA minigenes). The tRNA minigene that contains both intragenic control regions supports in vitro RNA synthesis in Xenopus laevis oocyte and HeLa cell transcription systems. The mutant which has deletions to nucleotide 7 within the mature tRNA coding region, pArg5.7, and minigenes derived from it do not support RNA synthesis in a Drosophila Kc cell transcription system. Xenopus and Hela extracts transcribe pArg5.7 albeit at reduced levels compared to the wild-type gene. The tRNA minigene that contained only the D-control region was not able to support RNA synthesis in any of these three transcription systems. A mutant tRNA gene comprising the 3' half of the tRNAArg gene similarly was not able to support RNA synthesis. These experiments show that the DNA sequence from nucleotides 7-58, which contains both intragenic control regions of the tRNA gene, possesses sufficient information to initiate specific transcription by RNA polymerase III in Xenopus and HeLa systems. The transcription efficiency of this tRNA minigene however is reduced to about 20% the transcription level of the wild type tRNA gene. This lowered level of transcriptional efficiency results from deleting the ends of the native tRNA gene and its adjacent flanking sequences. The affects of deleting 5' sequences are most pronounced in the Drosophila transcription system.

The sequences of two cloned genes from Drosophila which hybridize with tRNALys5 are reported. One gene, in plasmid pDt39, has a sequence which corresponds to the sequence of tRNA. The other gene, in pDt59R, differs in three nucleotides pairs. Both plasmids are transcribed in vitro with extracts of Drosophila Kc cells to give full-sized tRNA precursors with four additional nucleotides at the 5'-end as well as truncated molecules containing 35 nucleotides. This premature termination occurs in a block of four T residues within the mature coding region. Sequences flanking the tRNA genes show little in common except for the blocks of five or more T-residues beyond the 3'-end of the gene. pDt39 hybridizes to 84AB on the polytene chromosomes of Drosophila and pDt59R hybridizes to 29A.

The nucleotide sequence of glutamate tRNA1 from Schizosaccharomyces pombe was determined to be pU-C-C-G-U-U-G-U-m1G-G-U-C-C-A-A-C-G-G-C-D-A-G-G-A-U-U-C-G-U-C-G-C-U-U-U*-C-A-C-C-G-A-C-G-G-G-A-G-m5C-G-G-G-G-T-psi-C-G-A-C-U-C-C-C-C-G-C-A-A-C-G-G-A-G-C-C-AOH. The sequence differs markedly from that of S. cerevisiae tRNAGlu. S. pombe glutamate tRNA1 can be aminoacylated by the homologous glutaminyl-tRNA synthetase as well as by the corresponding enzyme from S. cerevisiae.

The nucleotide sequence of Drosophila melanogaster lysine tRNA2 was determined to be: pG-C-C-C-G-G-C-U-A-m2G-C-U-C-A-G-D-C-G-G-D-A-G-A-G-C-A-psi-G-A-G-A-C-U-C-U-U-t6A-A-psi-C-U-C-A-G-G-m7G-D-C-G-U-G-G-G-Xm-U-C-G-m1A-G-C-C-C-C-A-C-G-U-U-G-G-G-C-G-C-C-A(OH). With minor differences in the state of modification of some nucleotides, the sequence is the same as that of lysine tRNA2b from rabbit liver.

We report the sequences of six S. pombe tRNA genes including two genes for tRNAArg, and one gene each for tRNAGlu, tRNAHis, tRNALys and tRNAPhe. All tRNA genes are found independently in the genome and represent individual transcription units. The gene for tRNALys has an 8 bp long intervening sequence which cannot base-pair with the tRNA anticodon. In vitro transcription studies indicate that all genes are faithfully transcribed in a yeast extract. Sequence comparison of the 5' flanking regions of the tRNA genes did not show significant homologies; however, they are very rich in AT base pairs.

The known methods of enzymatic phosphorylation with [(32)P]phosphate of the 3'- or 5'-hydroxyl group of an oligonucleotide have been applied to oligonucleotides derived from Mycoplasma tRNA(Phe). The fingerprints obtained by both methods are very similar to each other and to that of uniformly labelled tRNA. The sequence of some oligonucleotides was determined by partial digestion of the 3'-phosphorylated fragment with spleen phosphodiesterase and of the corresponding 5'-phosphorylated fragment with venom phosphodiesterase.

Genomic blots of yellow lupin (Lupinus luteus) DNA digested with restriction nucleases and probed with 32P-labelled Lupinus 5S RNA reveal that 5S DNA is organized as tandemly repeated sequences of one size class, 342 bp. The DNA is extensively methylated. Two cloned BamHI ribosomal repeats were sequenced, revealing sequence divergence within both the coding and spacer regions.

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.

We have sequenced two genomic clones for wheat alpha/beta-gliadin storage protein genes. Comparison with a known sequence reveals close homology between the three and confirms the previously suspected evolutionary relatedness of members of this gliadin family. The coding region can be divided into six domains. Two unusual structures were found within this region: (i) The P-boxes which are composed of 12 codons, six of which are for proline, that are tandemly repeated four or five times; and (ii) Two polyglutamine stretches which consist of 18-22 tandemly repeated glutamine codons in one case, and 7-28 in the second. Analysis of the P-box structures revealed that certain mutations were probably present in the hypothetical ancestral alpha/beta-gliadin gene prior to gene multiplication. None of the genes have introns. All of the genes appear to contain typical eukaryotic promoters and also possess the double polyadenylation signal of plants.

We have determined the nucleotide sequence of the major species of E.colitRNA Ser and of a minor species having the same GGA anticodon. These two tRNAs should recognize the UCC and UCU codons, the most widely used codons for serine in the highly expressed genes of E.coli. The two sequences differ in only one position of the D-loop. Neither tRNA has a modified adenosine in the position 3′-adjacent to the anticodon. This can be rationalized on the basis of a structural constraint in the anticodon stem and may be related to optimization of the codon-anticodon interaction. Conparison of all E.coli serine tRNAs (and that encoded by bacteriophage T 4) reveals characteristic (possibly functional) features. Evolutionary analysis suggests an eubacterial origin of the T 4tRNA Ser gene and the existences of a recent common ancestor for thetRNAGUCSerandtRNAGUCSergenes. © 1985 IRL Press Limited.

The effect of base changes at the fourth position from the 3'-terminus of Escherichia coli initiator tRNAMet has been studied to test the 'discriminator hypothesis' which proposed that the nucleotide in this position might have a role in the specificity of the aminoacylation reaction. E. coli initiator tRNA lacking the 3'-terminal tetranucleotide was prepared by partial digestion with S1 nuclease. To construct tRNA analogs with different bases in the fourth position this truncated tRNA was joined by RNA ligase to each of four chemically synthesized 2',3'-ethoxy-methylidene tetranucleotides pACCA(em), pCCCA(em), pGCCA(em), and pUCCA(em). In vitro aminoacylation studies showed that all four molecules accepted methionine, albeit with different Vmax values.

We have isolated four segments of Drosophila melanogaster DNA that hybridize to homologous initiator tRNAMet. Three of the cloned fragments contain initiator tRNA genes, each of which can be transcribed in vitro. The fourth clone, pPW568, contains an initiator tRNA pseudogene which is not transcribed in vitro by RNA polymerase III. The pseudogene is contained in a 1.15 kb DNA fragment. This fragment has the characteristics of dispersed repetitive DNA and hybridizes in situ to at least 30 sites in the Drosophila genome. The arrangement of the initiator tRNA genes we have isolated, is different to that of other Drosophila tRNA gene families. The initiator tRNA genes are not clustered nor intermingled with other tRNA genes. They occur as single copies within an approximately 415-bp repeat segment, which is separated from other initiator tRNA genes by a mean distance of 17 kb. In situ hybridization to polytene chromosomes localizes these genes to the 61D region of the Drosophila genome. Hybridization analysis of genomic DNA indicates the presence of 8-9 non-allelic initiator tRNA genes in Drosophila melanogaster.

E. coli C6 rel- met- cys- was cultured in a fully supplemented medium and in media lacking cysteine or methionine. tRNA isolated from the three cultures containted, respectively, a normal complement of modified nucleosides; a deficiency in thiolated nucleosides and a deficiency in methylated nucleosides. Both sulfur-deficient tRNA and methyl-deficient tRNA contained large amounts of N-6- (delta-2-isopentenyl) adenosine and small amounts of the 2-methylthio derivative. Methyl-deficient tRNA contained, in addition a large amount of a cytokinin active, differently modified nucleoside that is believed to be a sulfur derivative of N6-(delta-2-isopentenyl) adenosine. The structure of this compound is unknown. When methly-deficient tRNA and the precusor the tRNA-Tyr su3-+ A25 were enzymatically methylated in vitro, methyl groups were incorporated into derivatives of isopentenyladenosine. These results indicate that the biosynthesis of the 2-methylthio derivative of isopentenyladenosine may occur in a sequential manner, i.e., thiolation of isopentenyladenosine followed by methylation.

A wheat genomic library was screened with two synthetic oligonucleotides (24 and 25 bases in length) complementary to a partial cDNA clone encoding a glutenin gene [Thompson et al. (1983) Theor. Appl. Genet. 67, 87-96]. Glutenins are large molecular weight aggregated proteins of grain endosperm, and major determinants of bread making quality of wheat. Of the two clones obtained one was fully characterized. It contained the sequence of the high molecular weight subunit of glutenin. The amino acid sequence derived from the gene sequence reveals a mature protein (817 amino acids) with a highly repeated structure of two different motifs corresponding to the high glutamine (35.7%), glycine (20.1%) and proline (13.1%) content. The gene does not contain an intron, and possesses a typical eukaryotic promoter; the RNA initiation site is 25-30 bases downstream.

Cell-free extracts of Escherichia coli and Bacillus subtilis catalyzed the tRNA-dependent, RNase A-sensitive formation of δ-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.