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

In a poly(U)-programmed translation system, neomycin stimulates the misincorporation of tyrosine and of serine which, according to Thompson and Stone (Thompson, R.C. and Stone, P.J. (1977) Proc. Natl. Acad. Sci. USA. 74, 198–202), are normally rejected at an initial discrimination step during the binding of charged tRNAs to the ribosome. In contrast, streptomycin favors the misincorporation of isoleucine which is normally rejected at a subsequent GTP-dependent discrimination step, the so-called proofreading step. The labeling of the ribosome with $\text{N-}\text{ethylmaleimide}$ mimics the effect of streptomycin in that it stimulates the misincorporation of isoleucine but not of tyrosine or serine. This effect is correlated with the labeling of protein S18 but not with that of protein S1. These observations indicate that the sulfhydryl group of protein S18 is located within a ribosomal domain involved in the proofreading control of tRNA selection. Taking into account our previous results that streptomycin and neomycin perturb ribosomal areas around the sulfhydryl groups of proteins S18 and S1, respectively, we suggest that these antibiotics induce misreading by different mechanisms which are linked to such perturbations.

Chinese hamster V-79 cells were treated with metabolic inhibitors of DNA or protein synthesis for various intervals of time after exposure of 3.0 or 5.0 J · m⁻². After removal of the metabolic block(s) the rate of DNA synthesis was followed by measuring the incorporation of [¹⁴C]thymidine into acid-insoluble material. A 2.5 or 5.0 h incubation with cycloheximide or hydroxyurea was effective in delaying the onset of the recovery in the rate of DNA synthesis that normally becomes evident several hours after exposure to ultraviolet light. By using concentrations of cycloheximide or hydroxyurea that inhibit DNA synthesis by a similar amount (70%), but protein synthesis by vastly different amounts (95% for cycloheximide; 0% for hydroxyurea), it was apparent that the delay in recovery caused by the treatment of the cells with cycloheximide could be accounted for entirely by its inhibitory effect on DNA synthesis. This suggests that the recovery in DNA synthetic rates following exposure of V-79 cells to ultraviolet light does not appear to require de novo protein synthesis, and therefore does not appear to require the involement of an inducible DNA repair process.

The nucleotide sequence selectivity of histone binding has been measured by thermal denaturation of reconstituted nucleoproteins. When DNAs of different average base compositions competed for the binding of purified histone fractions during in vitro reconstitutions in the presence of salt and urea, a decreasing $\text{(A + T)-binding}$ preference was observed following the order $\text{H}\text{1 >}\text{H}\text{2}\text{B}\text{>}\text{H}\text{5 >}\text{H}\text{2}\text{A}\text{> [}\text{H}\text{2}\text{A}\text{+}\text{H}\text{2}\text{B}\text{] > [}\text{H}\text{2}\text{A}\text{+}\text{H}\text{2}\text{B}\text{+}\text{H}\text{3 +}\text{H}\text{4]}$, $\text{[}\text{H}\text{1 + (}\text{H}\text{2}\text{A}\text{+}\text{H}\text{2}\text{B}\text{+}\text{H}\text{3 +}\text{H}\text{4)}{}_2\text{]}$. Nucleoprotein complexes formed under conditions shown to yield more physiologically comparable nucleosome structures revealed a minimal $\text{(A + T)-binding}$ preference. These results suggest that homotypic and heterotypic histone interactions decreased the nucleotide sequence selectivity of nucleosome binding.

The crystal structure of 1-methyl-5-nitrouracil monohydrate exhibits a novel intercalation of water molecules between pyrimidine bases that are stacked 6.2 Å apart. There is no direct hydrogen bonding along individual stacks, but a water molecule in one stack is hydrogen-bonded to three neighboring bases from adjacent stacks. These three bases are located in the same plane as the water molecule to which they are hydrogen-bonded. This mode of hydrogen bonding leads to infinite planes of parallel sheets of bases and water molecules at layers y ≈ 0 and $\text{1}\text{2}$ with no inter-sheet hydrogen bonding. On the basis of this intercalation and our model building studies, it is suggested that it would be stereochemically possible for a water molecule to act as a ‘spacer’ and stabilize the DNA helix, should a base be turned outside due to dynamical fluctuations of the DNA helix or non-complementary base opposition. Crystals of the title compound are monoclinic, space group C2, with cell constants a = 14.676(1), b = 6.243(1), c = 8.363(1)A, β = 100.88(2)°, Z = 4. Using 950 diffractometer data, the crystal structure of 1-methyl-5-nitrouracil monohydrate was determined by a direct inspection of the (|E|² − 1) Patterson function and refined to an R of 0.062.

The kinetics of hydrogen-tritium exchange reactions have been followed, using a Sephadex technique, for a double-helical $\text{poly(ribo}\text{-N}{}^6\text{-}\text{methyladenylic acid)-poly(ribouridylic acid)}$ complex. Only one (but not two) hydrogen in every $\text{N}{}^6\text{-}\text{methyladenine}\text{·}\text{uracil}$ basepair has been found to exchange at a measurably slow rate (1.2 · 10⁻³ s⁻¹) at 0°C. Thus, the proton exchange between the adenine-amino and uracil-imide groups in a double-helical polynucleotide is considered to be not as fast as has been suggested previously (approx. 10 s⁻¹) for an adenine · uracil basepair in a monomer system in a non-polar solvent.

We reported previously that Epstein-Barr (EB) virions and detergent-treated nucleocapsids co-purified with significant amounts of DNA polymerase activity that did not resemble other known host or viral polymerases. We report here that this species of DNA polymerase activity is present at early times after infection in lymphocytes abortively lytically infected (superinfected) with EB virus. However, studies with [³⁵S]methionine labeling suggest de novo synthesis of enzyme has not occurred. Conversely, drug-stimulated lymphocytes that synthesize EB viral late proteins and virions contain this species of polymerase to the virtual exclusion of all others. This EB viral polymerase shows a marked preference for nicked and gapped double-standed rather than primed single-stranded DNA templates. Its processiveness as measured on primed øX174 phage DNA template is lower than that of lymphocyte β polymerase. The data reported here are consistent with the hypothesis that the EB virion-associated DNA polymerase is synthesized at late times in the viral life cycle as are other structural proteins but it plays an important role early after viral infection. It is known that mature herpes virion DNA (including that of EB virus) is nicked and gapped and we propose that this virion polymerase repairs the viral DNA at an early stage in infection before viral DNA replication begins.

Proteins of membrane-bound ribosomes from normal liver contained 60–70% more phosphate than did proteins from free ribosomes. This difference was not a reflection of the phosphate contents of respective 40 S subunits. Instead, it was owing to the presence of high levels of phosphorylated proteins in the 60 S subunits, i.e., phosphate contents equal to or greater than those for 40 S subunits. The proteins of membrane-bound 60 S subunits contained twice the phosphate as free 60 S subunits. In regenerating rat liver, membrane-bound ribosomes had increased phosphate in the proteins of the 40 S subunits and decreased phosphate in proteins of the 60 S subunit when compared to controls from normal rat liver. No significant changes occurred in the proteins of free ribosomes from regenerating rat liver. These findings are discussed with respect to (a) the importance of assessing total phosphate contents of proteins in the study of ribosomal protein phosphorylation, and (b) the possible involvement of ribosomal protein phosphorylation in the segregation of ribosomes into free and membrane-bound populations and the regulation of these distributions to meet changes in the translational demands of the cell.

Spectroscopic studies on electronic structures were carried out for sulfur-containing nucleic acid bases, 2-thiouracil, 4-thiouracil, 2,4-dithiouracil and 2-thiocytosine along with their nucleosides. The absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra were interpreted in terms of tautomerism to suggest that 4-thiouracil is in a 2-keto-4-thione form and the other bases exist as an equilibrium mixture of thione and thiol forms in solution. The π-π★ transition energies of the thione structures of 2-thiouracil, 2-thiocytosine and their nucleosides are almost the same as those of unsubstituted pyrimidine bases and nucleosides. The n-π★ transition from a 2-thione function was clearly detected as a negative CD band at about 320 nm. On the other hand, from the absorption, CD and MCD spectra, the transitions of the 4-thiouracil derivatives were found to take place in an energy region lower than those of the corresponding parent bases.

We describe a re-examination of the cell-free protein synthetic activity of eukaryotic ribosomes having proteins phosphorylated to different extents. Ribosomal 40 S subunits were isolated both from a variety of cells in which there is relatively little phosphorylation of ribosomal protein S6, and from cells subjected in vivo to different stimuli that promote the extensive phosphorylation of protein S6. The ability of these subunits to bind Met-tRNA as well as the second amino acyl-tRNA (Val-tRNA) was compared in the presence of highly purified initiation factors, elongation factor EF-1 at various concentrations of 60 S subunits, 9 S globin mRNA and potassium ions. The ability of the subunits to synthesize polyphenylalanine was also studied using highly purified elongation factors. In no case was any significant difference in activity observed between ribosomes with protein S6 phosphory-lated to different extents. Similar, though less extensive, studies were performed comparing 60 S ribosomal subunits differing in the extent of phosphorylation of the acidic phosphoprotein, Lψ, and of L14. No difference in activity was observed between these ribosomes.

Covalently closed-circular, superhelical SV40 DNA was used in all experiments. EcoRI endonuclease- and HpaII enonuclease-generated unit-length linear duplex DNAs were digested with S₁ endonuclease under the conditions where single-stranded DNA was completely converted into the acid-soluble form. These were subjected to an end-to-end joining test with T4 DNA ligase. The ligation efficiency was significantly lower than that of the flush-ended linear duplex DNAs which were generated by both HpaI endonuclease digestion and the matching up of EcoRI-generated sticky end with Escherichia coli DNA polymerase I (Klenow fraction). However, the ligation efficiency of the S₁-treated DNAs increased up to same level as the flush-ended DNA upon treatment with E. coli DNA polymerase I. Similar results were obtained in the case of S₁-generated unit-length linear duplex DNA. S₁ does cleave both strands of superhelical DNA at unbasepaired sites.