Isolation and characterization of mutant bacteriophage T7 RNA polymerases.

We have isolated and characterized a number of bacteriophage T7 RNAP (RNA polymerase) null mutants. Most of the mutants found to be completely inactive in vitro map to one of the well-conserved blocks of residues in the family of RNAPs homologous to T7 RNAP. The in vitro phenotypes of a smaller number of partially active T7 RNAP mutants, mapping outside these well-conserved regions, support the following assignment of functions in T7 RNAP: (1) the N-terminal region of T7 RNAP contains a nascent RNA binding site that functions to retain the nascent chain within the ternary complex; (2) the region surrounding residue 240 is involved in binding the initiating NTP; (3) residues at the very C terminus of T7 RNAP are involved in binding the elongating NTP.     

Inorganic pyrophosphatase activity of the mouse spleen in the immune response and after treatment with bis-phosphonates

The inorganic pyrophosphatase activity was determined in different tissues of mice. The immunization of mice by sheep erythrocytes increased the inorganic pyrophosphatase activity of the spleen. The in vivo administration of bisphosphonates (40 mg per 1 g of mass), which are structural analogs of inorganic pyrophosphate (methylene bisphosphonic acid--MBPA, hydroxyethylidene bisphosphonic acid--HEBPA and aminomethylene bisphosphonic acid--AMBPA), inhibited the inorganic pyrophosphatase activity only by MBPA in the thymus and spleen but not in liver. The addition of MBPA, HEBPA as well as of phosphonoacetic acid, imidobisphosphate, bis(phosphonomethyl)-phosphonic acid, MBPA and phosphoric acid monoanhydride to cytosol from the mouse spleen led to the competitive (relative to the [Mg (PPi)2-] complex) inhibition of the inorganic pyrophosphatase activity. AMBPA didn't possess the analogous effect.     

Factors influencing the pulse character of RNA elongation in vitro by E. coli RNA polymerase

Pause location along primary structure of two RNA fragments each 200 nucleotide residues in the length synthesized from A1 promoters of T7 phage DNA and delta D111 T7 phage DNA was analyzed. No correlation between the location of pauses and GC-rich or self complementary regions of RNA were found. The location of pauses does not change upon the variation of the temperature or ionic strength. Concurrent variation of all four NTP concentrations also did not influence pausing pattern. However the distribution of pauses depends highly on the ratio of the individual substrate concentrations. Substitution of GTP by ITP changes the pausing pattern completely. Inorganic pyrophosphate (PPi) of inhibits RNA elongation preferentially in the regions: NAUN, CGUAG. The study of PPi action on RNA terminated with 3' OCH3-NMP suggest that the sequence-specific inhibition of RNA elongation may be a result of pyrophosphorolysis of terminal nucleotide residues of RNA. It was proposed that the pulse character of RNA elongation stems rather from differences in the kinetic constants of nucleotides attachment and pyrophosphorolysis from the 3'-termini of RNA than by termination signals encoded in the primary structure of DNA. The stable location of pauses in certain short oligonucleotides: AUG, AUU, AAU and some others is in favour of the hypothesis.     

Substrate properties of C′-methyl UTP derivatives in T7 RNA polymerase reactions. Evidence for N-type NTP conformation

The number of synthetic UTP analogues containing methyl groups in different positions of the ribose moiety were tested as substrates for T7 RNA polymerase (T7 RNAP). Two of these compounds (containing substituents in the 5′ position) were shown to be weak substrates of T7 RNAP. 3′Me-UTP was neither substrate nor inhibitor of T7 RNAP while 2′Me-UTP was shown to terminate RNA chain synthesis. Conformational analysis of the analogues and parent nucleotide using the force-field method indicates that the allowed conformation of UTP during its incorporation into the growing RNA chain by T7 RNAP is limited to the χ angle range of 192–256° of N-type conformation.     

The study of nucleoside triphosphate-binding center of DNA-dependent RNA-polymerase of phage T7 using GTP analogs

The NTP binding site of bacteriophage T7 DNA-dependent RNA polymerase was studied using GTP analogs. For four analogs the irreversible inhibition was demonstrated. The kinetic parameters for competitive (Ki) and irreversible (KI and k3) inhibition were determined. One of the analogs, 5'[2-hydroxy(4-iodoacetamido)benzoyl]guanosine, was shown to inactivate the enzyme rapidly due to the modification of SH-groups. Some suggestions on the structure of the RNA polymerase active site have been made.     

Interaction of dNTP-binding sites of human DNA polymerase alpha and The Klenow fragment of Escherichia coli DNA polymerase I with nucleotides, pyrophosphate and their analogs

AMP and NaF each taken separately were shown to activate DNA polymerization catalyzed by Klenow fragment of DNA polymerase I by means of interaction of AMP or NaF with 3'----5'-exonuclease center of the enzyme. In the presence of NaF which is a selective inhibitor of 3'----5'-exonuclease center, AMP is an inhibitor of polymerization competitive with respect to dATP. Ki values and the pattern of inhibition with respect to dATP were determined for AMP, ADP, ATP, carboxymethylphosphonyl-5'-AMP, Pi, PPi, PPPi, methylenediphosphonic acid and its ethylated esters, phosphonoformic acid, phosphonoacetic acid and its ethylated esters as well as for some bicarbonic acids in the reactions of DNA polymerization catalyzed by Klenow fragment of DNA polymerase I (in the presence of NaF) and DNA polymerase alpha from human placenta in the presence of poly(dT) template and r(pA)10 primer. All nucleotides and their analogs were found to be capable of competing with dATP for the active center of the enzyme. Most of the analogs of PPi and phosphonoacetic acid are inhibitors of Klenow fragment competitive with respect to dATP. Nowever these analogs display a mixed-type inhibition in the case of human DNA polymerase alpha. We postulated a similar mechanism of interaction for dNTP with both DNA-polymerases. It is suggested that each phosphate group of PPi makes equal contribution to the interaction with DNA polymerases and that the distance between the phosphate groups is important for this interaction. beta-phosphate of NTP or dNTP is suggested to make negligible contribution to the efficiency of the formation of enzyme complexes with dNTP. beta-phosphate is likely to be an essential point of PPi interaction with the active center of proteins during the cleavage of the alpha-beta-phosphodiester bond of dNTP in the reaction of DNA polymerization.     

Kinetics of inhibition of acetylcholinesterase by spin labeled acetylcholine analogs.

A series of spin labeled acetycholine analogs, in which the number of methylene groups between the quaternary nitrogen and the alcohol oxygen ranged between 1-5, have been examined as inhibitors of electric eel acetylcholinesterase. Evidence is presented suggesting that inhibition of acetylocholinesterase by the spin labeled ACH analogs is due to the high affinity of these compounds for the enzyme, inhibition is competitive and reversible. It has been shown that complex formation is of major importance in the reaction between spin labeled ACH analogs and acetylcholinesterase. The acetylation step has been shown to occur by demonstrating that the leaving group is released as the reaction proceeds. Complex formation has been demonstrated by means of kinetic criteria. Kinetic parameter have been measured for the five compounds, and correlations with alkaline hydrolysis are disussed.     

Binding of ADP and ATP analogs to cross-linked and non-cross-linked acto X S-1

We previously determined the binding constants of ADP, adenylyl imidodiphosphate (AMP-PNP), and inorganic pyrophosphate (PPi) to acto . myosin subfragment 1 (acto X S-1) by measuring the dissociation of acto X S-1 as a function of ATP analog concentration (Greene, L.E., and Eisenberg, E. (1980) J. Biol. Chem. 255, 543-548). In the present study, we reinvestigated this question by measuring the extent to which these ATP analogs inhibit the acto X S-1 ATPase activity using both cross-linked actin X S-1 and non-cross-linked proteins. No significant difference was found between the cross-linked and non-cross-linked acto X S-1 complexes in their affinity for either ADP or AMP-PNP. The binding constant of ADP to acto X S-1 determined by the inhibition method was in excellent agreement with that obtained previously by the dissociation method, both techniques giving values of about 7 X 10(3) M-1. However, this was not the case for AMP-PNP and PPi, with the inhibition method giving about 10-fold weaker binding constants than those determined previously by the dissociation method. Upon redoing our dissociation experiments over a wider range of actin concentrations than we used previously, we now find that the dissociation method gives much weaker values for the binding constants of PPi and AMP-PNP to acto X S-1, i.e. values on the order of 4 X 10(2) M-1. The very weak binding of these ATP analogs to acto X S-1 makes it difficult to obtain these values with great accuracy. Nevertheless, they seem to be in good agreement with the binding constants determined by the inhibition method. The weak binding of AMP-PNP and PPi to acto X S-1 is consistent with the recent fiber studies of Pate and Cooke (Pate, E., and Cooke, R. (1985) Biophys. J. 47, 773-780) and Schoenberg and Eisenberg (Schoenberg, M., and Eisenberg, E. (1986) Biophys. J. 48, 863-872).