[Unusual single-stranded DNA structures: extraordinarily thermo-stable mini-hairpin].
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Base Sequence↗
Biomedical subjects
I Hirao
Publications and source records attributed to I Hirao.
Higher-order structure of bovine mitochondrial tRNA(SerUGA): chemical modification and computer modeling.
On the basis of enzymatic probing and phylogenetic comparison, we have previously proposed that mammalian mitochondrial tRNA(sSer) (anticodon UGA) possess a slightly altered cloverleaf structure in which only one nucleotide exists between the acceptor stem and D stem (usually two nucleotides) and the anticodon stem consists of six base pairs (usually five base pairs) [Yokogawa et al. (1991) Nucleic Acids Res. 19, 6101-6105]. To ascertain whether such tRNA(sSer) can be folded into a normal L-shaped tertiary structure, the higher-order structure of bovine mitochondrial tRNA(SerUGA) was examined by chemical probing using dimethylsulfate and diethylpyrocarbonate, and on the basis of the results a tertiary structure model was obtained by computer modeling. It was found that a one-base-pair elongation in the anticodon stem was compensated for by multiple-base deletions in the D and extra loop regions of the tRNA(SerUGA), which resulted in preservation of an L-shaped tertiary structure similar to that of conventional tRNAs. By summarizing the findings, the general structural requirements of mitochondrial tRNAs necessary for their functioning in the mitochondrial translation system are considered.
Alkylating Agents↗
Nuclease resistance of an extraordinarily thermostable mini-hairpin DNA fragment, d(GCGAAGC) and its application to in vitro protein synthesis.
The nuclease resistance of a short, thermostable mini-hairpin, d(GCGAAGC), and other related hairpins was examined. Hairpins possessing a purine-rich (GAA) or (GAAA) loop appeared to be more resistant against nucleases than those with a pyrimidine-rich loop or single-stranded oligomers. Among 8 kinds of oligodeoxyribonucleotides examined, the fragment most resistant against nucleases was a hairpin with the sequence of d(CGCGAAGCG). This hairpin was then utilized for the stabilization of mRNA in an in vitro translation system; the 3'-terminal region of an mRNA was hybridized with an oligodeoxyribonucleotide including the sequence complementary to the 3'-terminus of the mRNA tagged with the nuclease-resistant d(CGCGAAGCG) hairpin sequence. By using this method, dihydrofolate reductase (DHFR) mRNA was stabilized against nucleases contaminating a cell-free translation system of E.coli, with a consequent increase in protein synthesis efficiency of 200%.
Base Sequence↗
Most compact hairpin-turn structure exerted by a short DNA fragment, d(GCGAAGC) in solution: an extraordinarily stable structure resistant to nucleases and heat.
The three-dimensional structure of a short DNA fragment, d(GCGAAGC) exhibiting an extraordinarily stable hairpin structure was determined by nuclear magnetic resonance spectroscopy. Two possible models were obtained by molecular modelling using distance and torsion constraints. Only one of these two models is the correct structure, which can clearly explain all the 1H chemical shifts. d(GCGAAGC) is folded back on itself between A4 and A5, and all the sugars in the fragment adopt the C2'-endo conformation. This compact molecule is stabilized by regular extensive base-stacking interaction within each B-form helical strand of G1C2G3A4 and A5G6C7, and by two G-C and one G3-A5 base pairs. The molecule is hard to differentiate into stem and loop regions, so that we classify it as a turn (hairpin-turn) structure experted by a single-stranded DNA. This highly stacked structure shows high thermostability and strong resistance against nucleases contained in E. coli extracts and in human serum.
Base Sequence↗
[Extraordinarily thermo-stable hairpin structures: partial structure of single-stranded nucleic acids].
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Base Sequence↗
Stabilization of mRNA in an Escherichia coli cell-free translation system.
It became clear that mRNA can be stabilized in a cell-free translation system of Escherichia coli by hybridization with a small DNA fragment at its 3' terminus. The stability increased when a small DNA fragment containing a stable hairpin structure with a GAAA loop was used. The enhancement of stabilization was brought about because the hairpin structure is resistant towards the nucleases contained in the translation system. The hairpin structure is effective by stabilizing the added DNA fragment itself towards the nucleases.
Amino Acid Sequence↗
Effect of tandem repeated AUG codons on translation efficiency of eukaryotic mRNA carrying a short leader sequence.
The effect on translation of multiple copies of the initiation codon AUG at the initiation site in a eukaryotic mRNA carrying a short leader sequence was tested in translation experiments in vitro. DNA, corresponding to a chimeric mRNA sequence consisting of the 5' leader region of brome mosaic virus (BMV) RNA4 and the goat pre-alpha-lactalbumin mRNA sequence, was prepared and transcribed in vitro using SP6 RNA polymerase. Site-directed mutagenesis was carried out to change the sequence around the initiation codon AUG. In a wheat germ translation system, the yield of protein obtained using the mRNA with a duplication of the AUG codons at the initiation site was 1.6 times that achieved when only one AUG was present. The rate of formation of the 80S initiation complex was measured by the ribosome binding assay using cycloheximide. A good correlation was observed between the ability to form the complex and translation efficiency.
Animals↗
Precise analyses of DNA structure by NMR.
Novel 1H NMR techniques were developed and applied to the analyses of the DNA structure. They distinguished the base proton signals of cytosine (uracil) from those of the other bases. Two dimensional experiments were also performed by using these techniques, and were found to be useful for signal assignments. Moreover, the (2'R)-[2'-2H]-labeled DNA 10-mer and 17-mer were synthesized to be used for the determination of precise structures. These stereoselective [2'-2H]-labeling made possible explicit stereospecific assignments and exact determination of the vicinal coupling constants, 3JHH, and thus the conformation of each deoxyribose ring was accurately determined.
Cytosine↗
Structural features and properties of an extraordinarily stable hairpin-turn structure of d(GCGAAGC).
The extraordinarily stable hairpin-like structure formed by a short DNA fragment, d(GCGAAGC), has been studied by NMR spectroscopy and UV melting behaviour. The fragment is folded back between A4 and A5, and forms two terminal G-C base pairs and a non-Watson-Crick G-A base pair. All the nucleotides adopt C2'-endo sugar puckers. Both G1C2G3A4 and A5G6C7 moieties have characteristics of B-form geometry and within each moiety all the bases are involved in extensive base-base interactions. Distortion from B-form occurs in only the three torsion angles between A4 and A5 residues, which causes a sharp turn in the structure. There is no clear distinction between a stem and a loop region, as observed in usual hairpin structures, so that we classify it as a turn (hairpin-turn) structure. In addition to the thermal stability, this fragment is more stable towards the attack of some nucleases than other single-stranded as well as usual hairpin DNA fragments.
Hot Temperature↗
Extraordinarily stable mini-hairpins: electrophoretical and thermal properties of the various sequence variants of d(GCGAAAGC) and their effect on DNA sequencing.
A small DNA fragment having a characteristic sequence d(GCGAAAGC) has been shown to form an extraordinarily stable mini-hairpin structure and to have an unusually rapid mobility in polyacrylamide gel electrophoresis, even when containing 7M urea. Here, we have studied the stability of the various sequence variants of d(GCGAAAGC) and the corresponding RNA fragments. Many such sequence variants form stable mini-hairpins in a similar manner to the d(GCGAAAGC) sequence. The RNA fragment, r(GCGAAAGC) also forms a mini-hairpin structure with less stability. The DNA mini-hairpins with GAAA or GAA loop are much more stable than DNA and RNA mini-hairpins with other loop sequence so far as has been examined. The stability difference between DNA and RNA mini-hairpins may be deduced to the stem structures formed by DNA (B form) and RNA (A form). The stable hairpins consisting of the GCGAAAGC sequence cause strong band compression on the sequencing gel. This phenomenon should be carefully considered in DNA sequencing.
Base Sequence↗
Stability difference between DNA and RNA mini-hairpins containing two G-C pairs.
The deoxyribonucleotide fragments, d(GCGAAAGC) and d(GCGAAGC) form extraordinarily stable mini-hairpin structures containing only two G-C pairs. In contrast, the corresponding ribonucleotide fragments, r(GCGAAAGC) and r(GCGAAGC) are not so stable, although they also form mini-hairpin structures. The stability difference between these DNA and RNA mini-hairpins was examined by NMR studies and the molecular mechanics calculations, and by comparing with the stability of several sequence variants. Their stability was deduced to vary delicately depending on helical patterns formed by DNA (B form) and RNA (A form) structures.
Base Composition↗
Relationship between the 5'-leader sequence of mRNAs and the translation efficiency in a eukaryotic in vitro system.
To examine the effect of the 5'-leader region of mRNAs in regulating translation efficiency, chimeric mRNAs which have various sequences of the 5'-leader were synthesized by the SP6 in vitro transcription system, and translated in the wheat germ in vitro translation system. Translation efficiency was affected by the length of the 5'-leader and the number of the codon A-U-G at the translation initiation site.
Animals↗
Highly stereoselective synthesis of (2'R)-[2'-2H]-deoxyribonucleosides and synthesis of their oligonucleotides.
Highly stereoselective synthesis of (2'R)-[2'-2H]-2'-deoxyribonucleosides (2'R:2'S = > 99:1) were accomplished by treating 2'-bromo-3',5'-O-TPDS-2'-deoxyribonucleosides with tributyltin deuteride at lower temperatures such as -60 degrees C in the presence of triethylborane. Moreover, synthesis of some oligodeoxyribonucleosides involving them will be described.
Boranes↗
A novel cloverleaf structure found in mammalian mitochondrial tRNA(Ser) (UCN).
Bovine mitochondrial tRNA(Ser) (UCN) has been thought to have two U-U mismatches at the top of the acceptor stem, as inferred from its gene sequence. However, this unusual structure has not been confirmed at the RNA level. In the course of investigating the structure and function of mitochondrial tRNAs, we have isolated the bovine liver mitochondrial tRNA(Ser) (UCN) and determined its complete sequence including the modified nucleotides. Analysis of the 5'-terminal nucleotide and enzymatic determination of the whole sequence of tRNA(Ser) (UCN) revealed that the tRNA started from the third nucleotide of the putative tRNA(Ser) (UCN) gene, which had formerly been supposed. Enzymatic probing of tRNA(Ser) (UCN) suggests that the tRNA possesses an unusual cloverleaf structure with the following characteristics. (1) There exists only one nucleotide between the acceptor stem with 7 base pairs and the D stem with 4 base pairs. (2) The anticodon stem seems to consist of 6 base pairs. Since the same type of cloverleaf structure as above could be constructed only for mitochondrial tRNA(Ser) (UCN) genes of mammals such as human, rat and mouse, but not for those of non-mammals such as chicken and frog, this unusual secondary structure seems to be conserved only in mammalian mitochondria.
Animals↗
Gel electrophoresis using a fluorescence agent for analysis and purification of non-labeled synthetic DNA fragments.
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DNA↗
Structure of mitochondrial tRNA.
Some mitochondrial (mt) tRNAs are presumed to have unusual secondary structures on the basis of DNA sequence analyses. However there are few tRNAs whose structures have been investigated at RNA level. Thus we isolated some mt tRNAs to investigated their structures and also synthesized mt tRNA by run-off transcription. In these experiments, following results have been obtained. 1) Bovine mt tRNA(Ser)(AGY) needs the interaction between the T-loop and D-arm for its aminoacylation activity. 2) The secondary structure of bovine mt tRNA(Ser)(UCN) is difficult from that predicted previously from its gene. 3) It has been confirmed by the hybridization using oligonucleotide probes that some Ascaris suum mt tRNAs possessing the unusual T-arms actually exist in its mitochondria.
Animals↗
Effect of hairpin DNA on poly(phenylalanine) synthesis.
In a poly(U) dependent poly(Phe) synthesis, an unaminoacylated tRNA(phe) binds to a ribosome and inhibits poly(Phe) synthesis in excess tRNA(Phe). A small DNA fragment having a hairpin structure was added to this system and was found to prevent an unaminoacylated tRNA(phe) from binding to a ribosome and enhance the efficiency of poly(Phe) synthesis in the presence of excess tRNA(phe).
Base Sequence↗
NMR spectroscopic study of single-stranded DNA fragments of d(CGGCGAAAGCCG) and d(CGGCAAAAGCCG).
We have investigated the structures of two kinds of single-stranded DNA fragments, d(CGGCGAAAGCCG) and d(CGGCAAAAGCCG), by use of NMR spectroscopy. It was found that the former takes a hair-pin like structure stabilized by hydrogen bonding of G/C base pairs in the stem region, while the latter takes a rather extended helical structure. The stable conformation of the former DNA is considered to originate from the stability of the sequence-specific conformation of the loop region rather than the stem region.
Base Sequence↗