Two-component ribonucleotidyl transferase from Escherichia coli. III. Effect of nucleoside diphosphates on synthesis and pyrophosphorolysis of polyribonucleotides by the enzyme.

1. The capacity of two-component ribonucleotidyl transferase to catalyze pyrophosphorolysis of polyribonucleotides is studied. 2. It is shown that nucleoside diphosphates (NDP), not being substrates for the enzyme, activate both the synthesis and pyrophosphorolysis of polynucleotides by the enzyme. The concentration of NDP is important for this effect: with an increase of NDP concentration the rate of synthesis increases and reaches a plateau at 10(-5) M NDP, while the rate of pyrophosphorolysis, attaining maximal values at 10(-5)--10(-3) M NDP, decreases with a further increase of NDP concentration. 3. The possible biological role of two-component ribonucleotidyl transferase is discussed.

Comparison of initiating abilities of primers of different length in polymerization reactions catalyzed by DNA polymerases from thermoacidophilic archaebacteria.

Optimal conditions for polymerization reaction catalyzed on poly(dA) and poly(dT) templates by DNA polymerases from thermoacidophilic archaebacteria--DNA polymerase A from Sulfolobus acidocaldarius and DNA polymerase B from Thermoplasma acidophilum--have been established. Values of Km and Vmax (60 degrees C) for a set of primers d(pA)n and d(pT)n have been estimated. Minimal primers for both enzymes are dNMP. Lengthening of primers by each mononucleotide increases their affinity about 2.16-fold. Linear dependence of log Km and of log vmax on the number of mononucleotide links in primers (n) has breaking point at n = 10. The value of Vmax is about 20% of that for decanucleotide. The affinity of the primer d(pA)9p(rib*) with a deoxyribosylurea residue at the 3'-end does not differ essentially from that of d(pA)9. Substitution of the 3'-terminal nucleotide of a complementary primer for a noncomplementary nucleotide, e.g., substitution of 3'-terminal A for C in d(pA)10 in the reaction catalyzed on poly(dT), decreases the affinity of a primer by one order of magnitude.

Nucleoside 5'-triphosphates modified at sugar residues as substrates for DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

The ability of a wide variety of nucleoside 5'-triphosphates with modified sugar moiety to serve as substrates in DNA synthesis catalyzed by DNA polymerase A from the archaebacterium Sulfolobus acidocaldarius was studied. Most of the dNTP analogs tested are shown to be specific terminating substrates for the synthesis irreversibly blocking further elongation of a nascent chain. The most powerful inhibitors were found to be 3'-amino derivatives of deoxy and arabino nucleoside triphosphates, while specific reverse transcriptase inhibitors, 3'-azido and 3'-methoxy derivatives of dNTP, were found to be inactive.

Electromechanical stability of planar lipid membranes from bipolar lipids of the thermoacidophilic archebacterium Sulfolobus acidocaldarius.

Stable planar membranes have been obtained from the bipolar lipid glycerol dialkyl nonitol tetraether (GDNT) extracted from the thermoacidophilic archebacterium Sulfolobus acidocaldarius. The electric capacity Cm, the resistance Rm and tension sigma of these membranes were measured. The dependence of the bipolar lipid membranes mean life time tau 1 on voltage was investigated. It was shown that the irreversible electric breakdown of membranes from GDNT and usual phospholipids is due to the same mechanism, viz., due to formation of a hydrophilic pore with an overcritical radius. Under electric field the GDNT molecules take U-shape, and the polar headgroups of such molecules cover the pore's interior.

Simple repetitive sequences in the genomes of archaebacteria.

Stretches of simple sequences poly(dG-dT).poly(dC-dA), poly(dG-dA).poly(dC-dT), poly(dG).poly(dC) and poly(dA).poly(dT), the occurrence of which is a characteristic feature of eukaryotic genomes, are found in the genomes of archaebacteria Halobacterium halobium and Sulfolobus acidocaldarius. In S. acidocaldarius these sequences constitute a considerable portion of the genome; they belong to a class of repetitive sequences dispersed throughout the genome, being transcribed and found in RNAs of different lengths.

A restriction endonuclease SuaI from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

A type II restriction endonuclease (SuaI) has been isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. The enzyme is an isoschizomer of BspRI. It does not cut S. acidocaldarius DNA, as the recognition sequence GGCC in this DNA contains modified nucleotide(s). The enzyme is most active at 60-70 degrees C and is highly thermostable.

[Molecular biology of archaebacteria]. / Molekuliarnaia biologiia arkhebakterii.

In the process of phylogenetic studies, based on the comparative analysis of sequences of 16S (18S) rRNA, C. Woese and collaborators discovered that some microorganisms, which previously had been described as bacteria, form a group named archaebacteria, differing from other bacteria as well as from eukaryotes to the same extent as the latter differ from each other. A review of the work leading to that result, as well as characteristics of archaebacteria with emphasis on their biochemistry and molecular biology, is presented.

A tw0-component ribonucleotidyl transferase from E. coli.

Some properties of an enzyme designated as a two component ribonucleotidyl transferase from E. coli are presented. The enzyme in the presence of magnesium ions catalyzes the synthesis of polyribonucleotide chains using all four nucleoside triphosphates as substrates. The enzyme consists of two components; component A in the presence of Mg2+ catalyzes the synthesis of homo- and heteropolymers using ATP, CTP and UTP but not GTP as substrates. Component B itself does not catalyze any synthesis at all, but its addition to component A affects this component in two ways: quantitatively- the activity of component A considerably increases, and qualitatively- both components together are capable of catalyzing the synthesis of polyribonucleotides consisting of all four ribonucleotides.

The genome of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius does not contain repetitive sequences.

Characteristics of genome organization in the sulfur-dependent thermoacidophilic archaebacterium Sulfolobus acidocaldarius have been studied. By means of hybridization analysis it is shown that the genome of S. acidocaldarius, unlike the genome of the extremely halophilic archaebacterium Halobacterium halobium, does not contain repetitive sequences.

[Isolation and properties of the SuaI restriction endonuclease from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius]. / Vydelenie i svoistva éndonukleazy restriktsii SuaI iz termoatsidofil'noi arkhebakterii Sulfolobus acidocaldarius.

A new restriction endonuclease SuaI was isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. The enzyme is an isoschizomer of BspR1; it recognizes tetranucleotide GGCC and cleaves DNA in the center of this sequence. SuaI requires Mg2+, the optimal concentration being 6 mM. KCl at concentrations above 25 mM significantly inhibits the enzyme activity. The pH optimum lies within the range of 6--7 at 70 degrees C, the temperature optimum is at 70--75 degrees C. The enzyme is highly stable at temperatures up to 80 degrees C. DNA of S. acidocaldarius is not cleaved by the enzyme.