Modeling the tertiary interactions in the eukaryotic selenocysteine tRNA.

A novel three-dimensional model of tertiary interactions in the core region of the eukaryotic selenocysteine tRNA is proposed based on the analysis of available nucleotide sequences. The model features the 7/5 tRNA(Sec) secondary structure characterized by seven and five base pairs in the acceptor and T-stems, respectively, and four nucleotides in the connector region between the acceptor and D-stems. The model suggests a unique system of tertiary interactions in the area between the major groove of the D-stem and the first base pair of the extra arm that provides a rigid orientation of the extra arm and contributes to the overall stability of the molecule. The model is consistent with available experimental data on serylation, selenylation, and phosphorylation of different tRNA(Sec) mutants. The important similarity between the proposed model and the structure of the tRNA(Ser) is shown. Based on this similarity, the ability of some tRNA(Ser) mutants to be serylated, selenylated, and phosphorylated was evaluated and found to be in a good agreement with experimental data.     

Unique secondary and tertiary structural features of the eucaryotic selenocysteine tRNA(Sec).

Cotranslational insertion of selenocysteine into selenoenzymes is mediated by a specialized transfer RNA, the tRNA(Sec). We have carried out the determination of the solution structure of the eucaryotic tRNA(Sec). Based on the enzymatic and chemical probing approach, we show that the secondary structure bears a few unprecedented features like a 9 bp aminoacid-, a 4 bp thymine- and a 6 bp dihydrouridine-stems. Surprisingly, the eighth nucleotide, although being a uridine, is base-paired and cannot therefore correspond to the single-stranded invariant U8 found in all tRNAs. Rather, experimental evidence led us to propose that the role of the invariant U8 is actually played by the tenth nucleotide which is an A, numbered A8 to indicate this fact. The experimental data therefore demonstrate that the cloverleaf structure we derived experimentally resembles the hand-folded model proposed by Böck et al (ref. 3). Using the solution data and computer modelling, we derived a three-dimensional structure model which shows some unique aspects. Basically, A8, A14, U21 form a novel type of tertiary interaction in which A8 interacts with the Hoogsteen sites of A14 which itself forms a Watson-Crick pair with U21. No coherent model containing the canonical 15-48 interaction could be derived. Thus, the number of tertiary interactions appear to be limited, leading to an uncoupling of the variable stem from the rest of the molecule.     

Antideterminants present in minihelix(Sec) hinder its recognition by prokaryotic elongation factor Tu.

During protein biosynthesis, all aminoacylated elongator tRNAs except selenocysteine-inserting tRNA Sec form ternary complexes with activated elongation factor. tRNA Sec is bound by its own translation factor, an elongation factor analogue, e.g. the SELB factor in prokaryotes. An apparent reason for this discrimination could be related to the unusual length of tRNA Sec amino acid-acceptor branch formed by 13 bp. However, it has been recently shown that an aspartylated minihelix of 13 bp derived from yeast tRNA Asp is an efficient substrate for Thermus thermophilus EF-Tu-GTP, suggesting that features other than the length of tRNA Sec prevent its recognition by EF-Tu-GTP. A stepwise mutational analysis of a minihelix derived from tRNA Sec in which sequence elements of tRNA Asp were introduced showed that the sequence of the amino acid- acceptor branch of Escherichia coli tRNA Sec contains a specific structural element that hinders its binding to T.thermophilus EF-Tu-GTP. This antideterminant is located in the 8th, 9th and 10th bp in the acceptor branch of tRNA Sec, corresponding to the last base pair in the amino acid acceptor stem and the two first pairs in the T-stem. The function of this C7.G66/G49.U65/C50.G64 box was tested by its transplantation into a minihelix derived from tRNA Asp, abolishing its recognition by EF-Tu-GTP. The specific role of this nucleotide combination is further supported by its absence in all known prokaryotic elongator tRNAs.     

Genes coding for the selenocysteine-inserting tRNA species from Desulfomicrobium baculatum and Clostridium thermoaceticum: structural and evolutionary implications.

The genes (selC) coding for the selenocysteine-inserting tRNA species (tRNA(Sec)) from Clostridium thermoaceticum and Desulfomicrobium baculatum were cloned and sequenced. Although they differ in numerous positions from the sequence of the Escherichia coli selC gene, they were able to complement the selC lesion of an E. coli mutant and to promote selenoprotein formation in the heterologous host. The tRNA(Sec) species from both organisms possess all of the unique primary, secondary, and tertiary structural features exhibited by E. coli tRNA(Sec) (C. Baron, E. Westhof, A. Böck, and R. Giegé, J. Mol. Biol. 231:274-292, 1993). The structural and functional properties of the tRNA(Sec) species from prokaryotes analyzed thus far support the notion that tRNA(Sec) may be an evolutionarily conserved structure whose function in the primordial genetic code was to decode UGA with selenocysteine.     

Selenocysteine inserting tRNAs: an overview

One of the recent discoveries in protein biosynthesis was the finding that selenocysteine, the 21st amino acid, is cotranslationally inserted into polypeptides under the direction of a UGA codon assisted by a specific structural signal in the mRNA. The key to selenocysteine biosynthesis and insertion is a special tRNA species, tRNA(Sec). The formation of selenocysteine from serine represents an interesting tRNA-mediated amino acid transformation. tRNA(Sec) (or the gene encoding it) has been found over all three domains of life. It displays a number of unique features that designate it a selenocysteine-inserting tRNA and differentiate it from canonical elongator tRNAs. Although there are still some uncertainties concerning the precise secondary and tertiary structures of eukaryal tRNA(Sec), the major identity determinant for selenocysteine biosynthesis and insertion appears to be the 13 bp long extended acceptor arm. In addition the core of the 3D structure of these tRNAs is different from that of class II tRNAs like tRNA(Sec). The biological implications of these structural differences still remain to be fully understood.     

Physical and biological features of polyoma virus mutants able to infect embryonal carcinoma cell lines

Three new polyoma mutants were selected for their ability to grow on the embryonal carcinoma cell line F9. These mutants share in common an insertion of two nucleotides, a thymine and an adenine, in the noncoding region located on the late side of the origin of replication. We have found that these insertions exist in all of the other polyoma virus mutants able to grow on F9 cells (Fujimura et al., Cell 23:809-814, 1981; Katinka et al., Nature (London) 290:720-722, 1981; K. Sekikawa and A. J. Levine, Proc. Natl. Acad. Sci. U.S.A. 78:1100-1104, 1981). The region containing these insertions could be folded into a stable secondary structure which included a guanine plus cytosine (G + C)-rich stem. The adenine and thymine were inserted in such a way that they maintained the palindrome in the G + C-rich stem and were complementary in the putative secondary structure that we present here. Another class of polyoma virus mutants selected on a multipotential carcinoma cell line (PCC4-Aza) were characterized by a more complex rearrangement (deletion and duplication) which occurred in the same region. This arrangement preserved the G + C-rich palindrome and also yielded a sequence which still allowed the folding of another type of stable secondary structure. The significance of these findings is discussed.     

Secondary structure and position of the cell-penetrating peptide transportan in SDS micelles as determined by NMR

Transportan is a 27-residue peptide (GWTLN SAGYL LGKIN LKALA ALAKK IL-amide) which has the ability to penetrate into living cells carrying a hydrophilic load. Transportan is a chimeric peptide constructed from the 12 N-terminal residues of galanin in the N-terminus with the 14-residue sequence of mastoparan in the C-terminus and a connecting lysine. Circular dichroism studies of transportan and mastoparan show that both peptides have close to random coil secondary structure in water. Sodium dodecyl sulfate (SDS) micelles induce 60% helix in transportan and 75% helix in mastoparan. The 600 MHz (1)H NMR studies of secondary structure in SDS micelles confirm the helix in mastoparan and show that in transportan the helix is localized to the mastoparan part. The less structured N-terminus of transportan has a secondary structure similar to that of the same sequence in galanin [Ohman, A., et al. (1998) Biochemistry 37, 9169-9178]. The position of mastoparan and transportan relative to the SDS micelle surface was studied by adding spin-labeled 5-doxyl- or 12-doxyl-stearic acid or Mn2+ to the peptide/micelle system. The combined results show that the peptides are for the most part buried in the SDS micelles. Only the C-terminal parts of both peptides and the central segment connecting the two parts of transportan are clearly surface exposed. For mastoparan, the secondary chemical shifts of the amide protons were found to vary periodically and display a pattern almost identical to those reported for mastoparan in phospholipid bicelles [Vold, R., et al. (1997) J. Biomol. NMR 9, 329-335], indicating similar structures and interactions in the two membrane-mimicking environments.     

Structure and evolution of a mouse tRNA gene cluster encoding tRNAAsp, tRNAGly and tRNAGlu and an unlinked, solitary gene encoding tRNAAsp

We have sequenced mouse tRNA genes from two recombinant lambda phage. An 1800 bp sequence from one phage contains 3 tRNA genes, potentially encoding tRNAAsp, tRNAGly, and tRNAGlu, separated by spacer sequences of 587 bp and 436 bp, respectively. The mouse tRNA gene cluster is homologous to a rat sequence (Sekiya et al., 1981, Nucleic Acids Res. 9, 2239-2250). The mouse and rat tRNAAsp and tRNAGly coding regions are identical. The tRNAGlu coding regions differ at two positions. The flanking sequences contain 3 non-homologous areas: a c. 100 bp insertion in the first mouse spacer, short tandemly repeated sequences in the second spacers and unrelated sequences at the 3' ends of the clusters. In contrast, most of the flanking regions are homologous, consisting of strings of consecutive, identical residues (5-17 bp) separated by single base differences and short insertions/deletions. The latter are often associated with short repeats. The homology of the flanking regions is c. 75%, similar to other murine genes. The second lambda clone contains a solitary mouse tRNAAsp gene. The coding region is identical to that of the clustered tRNAAsp gene. The 5' flanking regions of the two genes contain homologous areas (10-25 bp) separated by unrelated sequences. Overall, the flanking regions of the two mouse tRNAAsp genes are less homologous than those of the mouse and rat clusters.     

The length of the aminoacyl-acceptor stem of the selenocysteine-specific tRNA(Sec) of Escherichia coli is the determinant for binding to elongation factors SELB or Tu

Mutations in selC, which reduce the 8-base pair aminoacyl-acceptor helix to the canonical 7-base pair length (tRNA(Sec)(delAc] or which replace the extra arm of tRNA(Sec) by that of a serine acceptor tRNA species (tRNA(Sec)(ExS), block the function in selenoprotein synthesis in vivo (Baron, C., Heider, J., and B?ck, A. (1990) Nucleic Acids Res. 18, 6761-6766). tRNA(Sec), tRNA(Sec)(delAc), and tRNA(Sec)(ExS) were purified and analyzed for their interaction with purified seryl-tRNA synthetase, selenocysteine synthase and translation factors SELB and EF-Tu. It was found that seryl-tRNA synthetase displays 10-fold impaired Km and Kcat values for tRNA(Sec) in comparison to tRNA(Ser), decreasing the overall charging efficiency (Kcat/Km) of tRNA(Sec) to 1% of that characteristic for tRNA(Ser). tRNA(Sec)(ExS) was a less efficient substrate for the enzyme (Kcat/Km 0.2% of the tRNA(Ser) value) whereas the tRNA(Ser)(delAc) variant was charged with an approximately 2-3-fold improved rate compared to wild-type tRNA(Sec). Both mutant tRNA variants, when charged with L-serine, were able to interact with selenocysteine synthase to give rise to selenocysteyl-tRNA with tRNA(Sec)(ExS) being as efficient as wild-type tRNA(Sec). Seryl-tRNA(Sec)(delAc), on the other hand, was selenylated very slowly. Reduction of the length of the aminoacyl-acceptor stem to 7 base pairs prevented the interaction with translation factor SELB but allowed binding to EF-Tu, irrespective of whether tRNA(Sec)(delAc) was charged with serine or selenocysteine. The aminoacyl-acceptor helix of tRNA(Sec), therefore, is a major determinant directing binding to SELB and precluding interaction with EF-Tu.     

A correlation between N2-dimethylguanosine presence and alternate tRNA conformers.

Even though the evolutionary conservation of the cloverleaf model is strongly suggestive of powerful constraints on the secondary structure of functional tRNAs, some mitochondrial tRNAs cannot be folded into this form. From the optimal base pairing pattern of these recalcitrant tRNAs, structural correlations between the length of the anticodon stem and the lengths of connector regions between the two helical domains, formed by the coaxial stacking of the anticodon and D-stems and the acceptor and T-stems, have been derived and used to scan the tRNA and tRNA gene database. We show here that some cytosolic tRNA gene sequences that are compatible with the cloverleaf model can also be folded into patterns proposed for the unusual mitochondrial tRNAs. Furthermore, the ability to be folded into these atypical structures correlates in the mature RNA sequences with the presence of dimethylguanosine, whose role may be to prevent the unusual mitochondrial tRNA pattern folding.     

Structural analysis of Arabidopsis thaliana chromosome 3. I. Sequence features of the regions of 4,504,864 bp covered by sixty P1 and TAC clones.

Based on the physical map of Arabidopsis thaliana chromosome 3 previously constructed with CIC YAC, TAC, P1 and BAC clones (Sato, S. et al., DNA Res., 5, 163-168, 1998), a total of 60 P1 and TAC clones were sequenced, and the sequence features of the resulting 4,504,864 bp regions were analyzed by applying various computer programs for similarity search and gene modeling. As a result, a total of 1054 potential protein-coding genes were identified. The average density of the genes identified was 1 gene per 4066 bp. Introns were observed in 77% of the genes, and the average number per gene and the average length of the introns were 3.9 and 156 bp, respectively. These sequence features are essentially identical to those of chromosome 5 in our previous reports, but the gene density was slightly higher than that observed for chromosomes 2 and 4. The regions also contained 10 tRNA genes when searched by similarity to reported tRNA genes and the tRNA scan-SE program. The sequence data and information on the potential genes are available through the World Wide Web database KAOS (Kazusa Arabidopsis data Opening Site) at http://www.kazusa.or.jp/kaos/.     

The mitochondrial genome of the predatory mite Metaseiulus occidentalis (Arthropoda: Chelicerata: Acari: Phytoseiidae) is unexpectedly large and contains several novel features

The complete mitochondrial genome of the phytoseiid Metaseiulus occidentalis (Arthropoda: Chelicerata: Acari: Phytoseiidae) has been sequenced. It is 24,961 bp in length and contains a 14,695-bp unique region, a 345-bp triplicated region, and a 9921-bp duplicated region, in that order. The A+T content of the unique region is 76.9% and contains 11 protein coding (COI-III; ATP6-8; CytB; ND1, 2, 4, 5 and 4L), two ribosomal RNA (srRNA and lrRNA), 22 transfer RNA (tRNA) genes, and two copies of D-loop control sequence. Two genes (ND3 and 6) appear to be missing, but there is a large intergenic spacer (390 bp) present, which could contain ND3 if a different codon usage is employed. The gene order is completely different from the pattern in all other known chelicerates, including the horseshoe crab Limulus polyphemus [Lavrov et al., Mol. Biol. Evol., 2000; 17:813-824]. All the inferred tRNA genes are missing the TPsiC arm, but this arm has fused with the variable arm to generate a TV replacement loop. The duplicated region (9921 bp) contains 18 genes in the same order as in the unique region from CytB to tRNA-His, plus one copy of D-loop control sequence (311 bp) and a partial tRNA-Leu2 sequence (34 bp). The small triplicated region (345 bp) contains a D-loop control sequence (311 bp) and a partial tRNA-Leu2 sequence (34 bp). Because of these anomalies, amplifying sequences posed technical difficulties, but were accomplished by using a primer-walking strategy and increasing the AT content to 75% in the high-fidelity PCR dNTP mix. This is the first phytoseiid mitochondrial genome to be completely sequenced and the largest (25 kb) detected from the Chelicerata.     

An IS4 transposition causes a 13-bp duplication of phage lambda DNA and results in the constitutive expression of the cI and cro gene products

We have examined the nature of the additional DNA present in lambda hyp- mutants (Eisen et al., 1982). This DNA is an IS4 element in orientation I, in the y region of bacteriophage lambda at nucleotide position 39,139 (see Moore et al., 1979). Our assignment is based on (i) the similarity in size derived from the PstI, AvaI, and HindII restriction pattern and (ii) the DNA sequence of both the left and right lambda-IS4 DNA junctions in phage lambda hyp15rev4. The IS4 integration event resulted in the duplication of 13 bp of lambda DNA in contrast to the 11- and 12-bp duplications previously observed at the sites of IS4 integrations elsewhere (Klaer et al., 1981).     

Identification of a new inducible nodulation gene in Azorhizobium caulinodans.

The narrow host range bacterial strain Azorhizobium caulinodans ORS571 induces the formation of nitrogen-fixing nodules on the root and stem of the tropical legume Sesbania rostrata. Here, a new flavonoid-inducible locus of ORS571 is described, locus 4. The locus was identified and isolated via the occurrence of particular sequences, the gamma and delta elements. These elements are reiterated in the ORS571 genome, linked to symbiotic loci. Sequencing of locus 4 showed the presence of an open reading frame (ORF6) that is flanked downstream by a gamma element and upstream by a delta element. The gamma element is approximately 180 bp in size, and shows homology to the insertion element ISRm3, an insertion sequence belonging to a distinct class of IS elements. The delta element is about 300 bp in size and has homology with repeated sequences found in other Rhizobiaceae. The ORF6 gene product shows a low, but significant homology to the mouse mastocytoma antigen P35B (Szikora et al., EMBO J. 9: 1041-1050, 1990) and to a class of NAD/NADP-binding sugar epimerase/dehydrogenases (Pissowotzki et al., Mol. Gen. Genet. 231: 113-213, 1991). Immediately upstream from ORF6, a nod box-related sequence is present, the arrangement of which is fully consistent with a recently presented model for the nod box structure (Goethals et al., Proc. Natl. Acad. Sci. USA 89: 1646-1650, 1992). Insertional inactivation of ORF6 did not affect the nodulation and fixation performance on S. rostrata. However, on S. formosa roots the nodulation kinetics of such a mutant was clearly affected (about 5 days delay). We propose to call this new symbiotic gene nolK.     

Mutagenesis of selC, the gene for the selenocysteine-inserting tRNA-species in E. coli: effects on in vivo function.

The selenocysteine-inserting tRNA (tRNA(Sec)) of E. coli differs in a number of structural features from all other elongator tRNA species. To analyse the functional implications of the deviations from the consensus, these positions have been reverted to the canonical configuration. The following results were obtained: (i) inversion of the purine/pyrimidine pair at position 11/24 and change of the purine at position 8 into the universally conserved U had no functional consequence whereas replacements of U9 by G9 and of U14 by A14 decreased the efficiency of selenocysteine insertion as measured by translation of the fdhF message; (ii) deleting one basepair in the aminoacyl acceptor stem, thus creating the canonical 7 bp configuration, inactivated tRNA(Sec); (iii) replacement of the extra arm by that of a serine-inserting tRNA abolished the activity whereas reduction by 1 base or the insertion of three bases partially reduced function; (iv) change of the anticodon to that of a serine inserter abolished the capacity to decode UGA140 whereas the alteration to a cysteine codon permitted 30% read-through. However, the variant with the serine-specific anticodon efficiently inserted selenocysteine into a gene product when the UGA140 of the fdhF mRNA was replaced by a serine codon (UCA). Significantly, none of these changes resulted in the non-specific incorporation of selenocysteine into protein, indicating that the mRNA context also plays a major role in directing insertion. Taken together, the results demonstrate that the 8-basepair acceptor stem and the long extra arm are crucial determinants of tRNA(Sec) which enable decoding of UGA140 in the fdhF message.