Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis最新文献

‘Strong-stop’ DNA, complementary to the 5′-terminal 101 nucleotides of Rous sarcoma virus genomic RNA and subgenomic mRNAs can be used to isolate RSV-specific mRNA from infected cells. To analyse leader sequences of these mRNAs and to study the mechanism of their translation, relatively large quantities of strong-stop DNA are required. The construction of four plasmids carrying useful strong-stop DNA sequences for mRNA isolation is described. One such hybrid plasmid carries a DNA insertion containing sequences derived from a region starting 42 nucleotides from the 5′-end of RSV RNA, and ending at the tRNA^Try primer attachment site. Since this plasmid lacks the 20-nucleotide terminal repeat sequence it can be used for the specific isolation of 5′-end fragments of RSV-specific mRNAs and of complementary DNA transcripts of these fragments.

The mechanism by which brusatol inhibits protein synthesis in rabbit reticulocytes has been investigated. When added to reticulocyte lysates, brusatol inhibits endogenous protein synthesis only after a lag of 2–4 min at 30°C. During this period 80 S ribosomes accumulate. Brusatol is equally effective in inhibiting endogenous protein synthesis in lysates and poly(U)-directed polyphenylalanine synthesis with runoff ribosomes. In fractionated reticulocyte systems, brusatol does not inhibit formation of the ternary, 40 S, and 80 S initiation complexes, but does inhibit the reaction of puromycin with initiation complexes containing [³⁵S]Met-tRNA_f. These data suggest that brusatol inhibits the peptidyl transferase elongation reaction of protein synthesis, but can do so only after one round of protein synthesis has been completed. Thus, the mechanism of action of brusatol in the rabbit reticulocyte system is very similar to the effects previously reported for bruceantin in a yeast system.

We have shown that pyridoxal 5′-phosphate is an effective inhibitor of Rauscher leukemia virus DNA polymerase (Biochemistry 15 (1976) 3620). Detailed studies of this inhibition revealed that, in addition to the phosphate and aldehyde groups of pyridoxal phosphate, the presence of a divalent cation is essential for the inhibitory action. The synthesis directed by template primers containing GC base-pairs exhibited more resistance to pyridoxal phosphate inhibition than did that directed by AT base-paired templates. Maximal inhibitory activity of pyridoxal phosphate, however, is noted in the presence of Mn²⁺, irrespective of which template-primer is used to direct the DNA synthesis. The action of pyridoxal phosphate on the substrate binding site may be deduced from the observations that: (a) only the substrate triphosphate is able to reverse the pyridoxal phosphate-mediated inhibition; (b) the inhibition kinetics exhibit a classical competitive pattern with the substrate; (c) analogous to substrate deoxynucleoside triphosphates, the inhibitor is also accepted only in the form of its divalent metal ion complex; and (d) substrate site-specific labeling of RLV DNA polymerase has been shown to occur by linking covalently the pyridoxal phosphate bound to a lysine residue at the substrate binding site.

The title compound (hereafter abbreviated as 6(R),5′-cyclo-hUrd) is synthesized from 2′,3′-O-isopropylidene-5′-deoxy-5′-iodouridine and its molecular structure has been determined by X-ray analysis. 6(R),5′-Cyclo-hUrd crystallizes in space group C2 with Z = 4, and unit-cell dimensions a = 11.220 (2), b = 6.393 (1), c = 18.963 (3)$\text{A}\text{̊}\text{and}\text{α = 107.98 (1)°}$. The structure was solved by direct interpretation of the three-dimensional Patterson function and refined to a final R index of 0.063. In the crystal the glycosyl torsion angle $\text{ч}{}_{\mathrm{<mtext>CN</mtext>}}$ is 60.7° (anti conformation) and the dihydrouracil ring adopts a half-chair conformation. The puckering of the ribose ring is an unusual O(1′)-exo (P = 267°, τ_m = 47°). The coupling constants of the ¹H-NMR spectrum measured in C²HCl3 solution indicate that the overall conformation of 6(R),5′-cyclo-hUrd found in crystal is also maintained in solution. The theoretical calculations of coupling constants by the finite perturbation theory (FPT) intermediate neglect of differential overlap, self-consistent field molecular orbital (INDO SCF-MO) method indicate that the deviation of the observed coupling constants of sugar ring protons from those predicted by applying modified Karplus-type formula to the X-ray structure could be due to the strains around the sugar ring carbon atoms attached by protons.

The control of protein synthesis by hemin in rabbit reticulocyte lysate is mediated by the formation of a high molecular weight protein inhibitor (or translational repressor) of polypeptide chain initiation from a precursor (prorepressor), which acts by phosphorylating the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 35 000 (α) subunit of eIF-2. We originally isolated a post-ribosomal supernatant factor from reticulocyte lysate, distinct from soluble eIF-2, that could completely reverse the inhibition of protein synthesis that occurs when reticulocyte lysate is incubated in the absence of hemin. We have found that this supernatant factor promotes the inactivation of the intermediate form of the translational repressor (generated within 1 h of incubation of the prorepressor in the absence of hemin), but only in the presence of GTP. This inactivation is not seen with the irreversible form of the translational repressor (generated after prolonged incubation of the prorepressor) and does not occur with other nucleoside triphosphates, dGTP or GDP. In addition, the inactivation reaction is not dependent upon Mg²⁺ and is not mediated by cyclic GMP. When lyate samples were pulsed with [γ-³²P]ATP at 15 min of incubation, the addition of the supernatant factor at zero time or after 7 min was associated with preventing or reversing the phosphorylation of eIF-2α. In contrast, another soluble reticulocyte protein termed reversing factor can be separated from the supernatant factor on phosphocellulose, partly overcomes the effect of the irreversible translational repressor, and is associated with increased phosphorylation of eIF-2α.

A cyclic GMP-dependent protein kinase was previously found in the 0.3 M NaCl extract of avian liver nucleoli [1]. The kinase phosphorylates preferentially a protein of a molecular weight of approximately 11 000 present in calf thymus histone mixture (type IIA, Sigma) and in isolated liver nucleoli. Further studies with purified protein substrates have now indicated that the chromatin-associated protein, which is preferentially phosphorylated by the cyclic GMP-dependent kinase, is high mobility group protein HMG 14. Histone H1 was also a relatively good phosphate acceptor but in this case the phosphorylation was not cyclic GMP-dependent and therefore due to a different protein kinase present in the partially purified nucleolar extract. Acid hydrolysis of the phosphorylated HMG 14 and subsequent analysis by chromatography and high-voltage electrophoresis indicated that the phosphorylated amino acid residue in HMG 14 is phosphoserine.

A pyrimidine nucleoside phosphorylase was partially purified from human blood platelets. The purified enzyme, as well as crude enzyme preparations, catalyses the phosphorolysis of thymidine and deoxyuridine, but not of uridine, and is able to catalyse direct pentosyl transfer from these deoxyribonucleosides to uracil or thymine; this enzyme has the properties of a thymidine phosphorylase. It has a molecular weight of about 110 000 and is composed of two identical subunits; it is phosphate dependent, has a maximal activity at a pH value of 5.7, and an isoelectric point of 4.4. This enzyme was mainly of cytoplasmic origin. Although platelet thymidine phosphorylase could promote the degradation or synthesis of thymidine, intact platelets degraded thymidine but were not able to synthesize thymidine from thymine. Blood platelets may play an important role in the degradation of plasma thymidine.

DNA polymerase α (EC 2.7.7.7) from calf thymus has been separated into three molecular species, i.e., 10 S DNA polymerase α, 6.5 S DNA polymerase α-1 and 6.5 S DNA polymerase α-2 (Masaki, S. and Yoshida, S. (1978) Biochim. Biophys. Acta 531, 74–88; Yoshida, S., Yamada, M., Masaki, S. and Seneyoshi, M. (1979) Cancer Res. 39, 3955–3958). Among these three, 10 S DNA polymerase α and 6.5 S DNA polymerase α-2 were found to copy efficiently poly(rA) · oligo(dT), a template-primer, which was thought to be specific for DNA polymerase γ or β. 6.5 S DNA polymerase α-1, however, could not use the ribopolymer as a template. The poly(rA) · oligo(dT)-dependent activities of DNA polymerase α species differed markedly from those with activated calf thymus DNA in sensitivity to various reagents: the former was inhibited more than 80% by 80 mM KCl, while the latter was stimulated somewhat. Furthermore, aphidicolin, a specific inhibitor of DNA polymerase α, did not inhibit the poly(rA) · oligo(dT)-dependent activity. 2′,3′-DideoxyTTp, a potent inhibitor of DNA polymerase β or γ, slightly inhibited the reactions with poly(rA) · oligo(dT), while it did not inhibit the reactions with activated DNA. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for dTTP on poly(rA) · oligo(dT) template were 260 and 70 μM for 10 S α and 6.5 S α-2, respectively; these values were much higher than those obtained on activated DNA template (8–10 μM).

The physiological androgen, 5α-dihydrotestosterone (DHT), enhances a progesterone-regulated protein (uteroglobin) synthesis in the rabbit uterus. In order to clarify the induction mechanism(s), rabbits were treated for 5 days with DHT alone or concomitantly with a nonsteroidal antiandrogen, RU 23908, or with different doses of estradiol. Uteroglobin content was measured in the uterine fluid by radioimmunoassay and uteroglobin mRNA activity in uterine tissues using cell-free translation in vitro. Uteroglobin induction elicited by DHT was inhibited by a small dose of estradiol, but not by antiandrogen. These results support the idea that androgens bring about their action on uteroglobin synthesis via a mechanism involving uterine progesterone receptor.

A nucleic acid helix-destabilizing protein has been purified from Saccharomyces cerevisiae using affinity chromatographic techniques. Crude protein extracts at low ionic strength (approx. 0.05 M) were applied sequentially to tandem columns of native DNA-cellulose, aminophenyl-phosphoryl-UMP-agarose, poly(I · C)-agarose, poly(U)-cellulose and denatured DNA-cellulose. The 2 M NaCl eluant of the poly(U)-cellulose column was dialyzed to low ionic strength and recycled through native DNA-cellulose, poly(I · C)-agarose and poly(U)-cellulose. Purified helix-destabilizing protein eluted from the poly(U)-cellulose between 0.1 and 0.5 M NaCl. On the basis of enzymatic activity, immunological cross-reactivity, mobility on SDS gels, amino acid analysis and preliminary peptide mapping experiments, this material was identified as an isozymic fraction of glyceraldehyde-3-phosphate dehydrogenase. The major crystallizable isozyme of this enzyme from yeast is, however, considerably more acidic than the helix-destabilizing protein, and displays significantly lower helix-destabilizing activity. Stoichiometric levels of the isolated protein at low (approx. 0.01) ionic strength depress the $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ of poly(A-U) and poly[d(A-T)] by as much as 28 and 22°C, respectively. Longer double helices, poly(A · U) and Clostridium perfringens DNA, are also denatured by the helix-destabilizing protein, but at relatively slow rates. The binding of this protein to [³H]-poly(U) on nitrocellulose filters is [Na⁺]-dependent, with a 50% reduction at 0.09 M NaCl. Based on its effect on the circular dichroism spectrum of poly(A), the protein was shown to distort the conformation of the polynucleotide chain. An analogous protein from mammalian cells, P8, was also shown to depress poly(A-U) $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$.