Structure of RecA-DNA complexes studied by combination of linear dichroism and small-angle neutron scattering measurements on flow-oriented samples.

By combining anisotropy of small-angle neutron scattering (SANS) and optical anisotropy (linear dichroism, l.d.) on flow-oriented RecA-DNA complexes, the average DNA-base orientation has been determined in RecA complexes with double-stranded (ds) as well as single-stranded (ss) DNA. From the anisotropy of the two-dimensional SANS intensity representation, the second moment orientation function S is obtained. Knowledge of S is crucial for the interpretation of l.d. spectra in terms of orientation of the DNA bases and the aromatic amino acid residues. The DNA-base planes are essentially perpendicular to the fibre axis of the complex between RecA and dsDNA in the presence of cofactor ATP gamma S. A somewhat tilted base geometry is found for the RecA-ATP gamma S complexes with single-stranded poly(dT) and poly(d epsilon A). This behaviour contrasts the RecA-ssDNA complex formed without cofactor which displays a poor orientation of the bases. Well-ordered bases in the ssDNA-RecA complex is possibly reflecting the role of RecA in preparing a nucleotide strand for base-pairing in the search-for-homology process. While the central SANS intensity is essentially independent of the pitch of the helical complex, a secondary intensity maximum, which becomes focused upon flow orientation, is found to be a sensitive measure of the pitch. The pitch values for the complexes compare well with cryo-electron microscopy results but are slightly larger than those seen for uranyl-stained samples.     

Binding of recA protein to Z-form DNA studied with circular and linear dichroism spectroscopy

Binding of RecA to poly(dG-m5dC) and poly(dG-dC) under B- and Z-form conditions was studied using circular dichroism (CD) and linear dichroism (LD). LD revealed a quantitative binding of RecA to Mg2+-induced Z-form poly(dG-m5dC) with a stoichiometry of 3.1 base pairs/RecA monomer, which is slightly larger than the 2.7 base pairs observed for the B-form. The LD spectra indicate a preferentially perpendicular orientation of DNA bases and a rather parallel orientation of the tryptophan residues relative to the fiber axis in both complexes. The association rate of RecA to Z-form DNA was found to be slower than to B-form. CD measurements showed that the polynucleotide conformation is retained upon RecA binding, and CD and LD confirm that RecA binds to both forms of DNA. The Mg2+-induced Z-form is shown to be retransformed into B-form, both in free and in RecA-complexed polynucleotides by addition of NaCl, whereas the B----Z transition cannot be induced by addition of Mg2+ when the polynucleotide is complexed with RecA. From this it is inferred that RecA does not stabilize the Z-conformation of the polynucleotide but that it can kinetically "freeze" the polynucleotide in its B-conformation. On all essential points, the same conclusions were also reached in a corresponding study of unmethylated poly(dG-dC) with the Z-form induced by Mn2+.     

Coordination and internal exchange of two DNA molecules in a RecA filament in the presence of hydrolysing ATP. Information on ATP-RecA-DNA structure from linear dichroism spectroscopy.

Solution structure of complexes between DNA and recombinase RecA from Escherchia coli, in the presence of the physiological cofactor ATP, is probed by flow linear dichroism (LD) spectroscopy. A problem of ADP accumulation which promotes dissociation of DNA-RecA is circumvented by using an ATP-regenerating system. The LD features indicate that the local structure of the complex is very similar to that found in the presence of the non-hydrolysable analog of ATP, adenosine-5'-O-[gamma-thio]triphosphate (ATP[gamma S]); the DNA bases are oriented with their planes preferentially perpendicular to the long axis of the filament, while the indole chromophores of the two tryptophan residues of RecA are rather parallel to this reference direction. A much smaller overall amplitude of the LD spectrum, compared to ATP[gamma S], is interpreted as a result of fast dissociation of RecA due to hydrolysis of ATP, producing transiently naked DNA regions which act like flexible joints, diminishing the macroscopic orientation of the RecA filaments. However, the ATP hydrolysis is not found to prevent simultaneous accommodation of two non-complementary DNA molecules in the RecA complex, as judged from the LD behaviour upon successive addition of two different polynucleotides or modified DNA strands. A notable difference from corresponding complexes formed with ATP[gamma S] is that, in the presence of ATP hydrolysis, the order in which the two DNA molecules have been added is insignificant as judged from virtually identical resulting structures; this observation indicates that exchange of DNA occurs between the two DNA accommodation sites within the RecA filament.     

Fibers of RecA protein and complexes of RecA protein and single-stranded phi X174 DNA as visualized by negative-stain electron microscopy

Monomers of purified RecA protein polymerize into helical fibers whose pitch is 7.2 nm to 7.5 nm and whose diameter is 11 nm. Either short (approximately 0.2 micron), single fibers, or bundles of aligned, longer fibers, can be formed preferentially, by varying the Mg2+ concentration. When RecA protein is bound to circular, single-stranded phi X174 DNA it forms helical fibers of different classes of contour lengths, ranging from 0.98 micron, depending upon the conditions of assembly. Two different helical pitches are found, one of 9.3 nm when the incubation buffer contains, besides the obligatory Mg2+, either ATP gamma S or ATP accompanied by single-strand binding protein, and one of 5.5 nm when the latter additives are omitted. Preformed fibers of the compact type can be converted to open ones of 9.3 nm pitch upon addition of ATP gamma S, even after the removal of unbound RecA. All signs of helicity are obliterated upon glutaraldehyde cross-linking except in those fibers whose assembly has been mediated by ATP gamma S. RecA protein and single-strand binding protein are competitively bound to single-stranded DNA. Composite complexes, however, are not encountered unless ATP gamma S is present. Otherwise, segments of DNA that are coated by one or the other protein are seen as separate regions. When the assembly of complexes of single-stranded DNA and RecA is mediated by single-strand binding protein and ATP, the axial separation between successive bases is 0 X 42 nm, somewhat greater than the axial distance between bases in one strand of duplex DNA in the B form. It is proposed that the bases of the single-stranded DNA in the complex are located near its inner surface, and that base-pairing with double-stranded DNA takes place following invasion of the central cavity of the complex.     

Cofactor-induced orientation of the DNA bases in single-stranded DNA complexed with RecA protein. A fluorescence anisotropy and time-decay study

The structure of the RecA-single-stranded DNA complex was investigated by studying the fluorescence emission of poly(deoxy-1,N6-ethenoadenylic acid (poly(d epsilon A)), a fluorescent derivative of poly(dA), under various viscosity conditions. The fluorescence intensity and average lifetime of poly(d epsilon A) are much smaller than those of nonpolymerized monoethenonucleotides (1,N6-ethenoadenosine 5'-triphosphate and 1,N6-ethenoadenine deoxyribose 5'-monophosphate) at low viscosity and reflect intramolecular base-base collisions in the polymer. They considerably increased upon RecA binding, both in the presence and absence of cofactor ATP or adenosine 5'-O-(3-thiotriphosphate). This increase, as well as the increase in fluorescence anisotropy upon RecA binding, was very similar to that which resulted from sucrose addition to free poly(d epsilon A). These observations point to a decrease in the mobility of DNA bases upon RecA binding. In the presence of cofactor, the fluorescence features became independent of viscosity. This strongly suggests the absence of base motion of significant amplitude on the time scale of the fluorescence lifetime (about 10 ns). In the absence of cofactor, however, these features remained sensitive to viscosity, implying residual local motions of the bases. Such cofactor-dependent rigid attachment of DNA bases to stiff phosphate backbone could facilitate the search for homology between two DNA molecules during recombination.     

Two forms of RecA-single-stranded DNA-adenosine 5'-O-(3-thiotriphosphate) complexes with different activities for cleavage of phage phi 80 cI repressor

The kinetics of cleavage of the phage phi 80 cI repressor by Escherichia coli RecA protein were studied. The rate of cleavage in the presence of single-stranded DNA (ssDNA) and either adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), ATP or dATP is very low in the first hour at 37 degrees C and then increases sharply as incubation continues. The initial rate of cleavage of the repressor is greatly increased by incubating the RecA protein with ssDNA prior to addition of ATP gamma S and the repressor. However, when ATP gamma S is present during preincubation of RecA protein with ssDNA, the stimulatory effect of preincubation is greatly reduced. This difference in the effect of preincubation in two different conditions can be explained by formation of RecA-ssDNA-ATP gamma S complexes with different activities for cleavage of the repressor. The active complex is formed by binding of ATP gamma S to a complex of RecA protein and ssDNA. However, when the RecA protein binds to ATP gamma S prior to its binding to ssDNA, the resulting complex has no or only very weak cleavage activity toward the repressor.     

Adenosine 5'-O-(3-thiotriphosphate) hydrolysis by dynein

The interaction of dynein with ATP gamma S, a phosphorothioate analogue of ATP, has been investigated in depth. The hydrolyses of ATP gamma S and of ATP were shown to be mutually competitive. ATP gamma S induced complete dissociation of the microtubule-dynein complex such that the time course of dissociation monitored by stopped-flow light-scattering methods followed a single exponential. The ATP gamma S concentration dependence of the rate of dissociation was hyperbolic, indicating that the dissociation is at least a two-step process: M.D + ATP gamma S in equilibrium M.D.ATP gamma S----M + D.ATP gamma S. The fit to the hyperbola gives an apparent Kd = 0.5 mM for the binding of ATP gamma S to the microtubule-dynein complex, and the maximal rate of 45 s-1 defines the rate of dissociation of the ternary M.D.ATP gamma S complex. Rapid quench-flow experiments demonstrated that the hydrolysis of ATP gamma S by dynein exhibited an initial burst of product formation. The size of the burst was 1.2 mol/10(6) g of dynein, comparable to that in the case of ATP hydrolysis. The steady-state rate of ATP gamma S turnover by dynein was activated by MAP-free microtubules. Because the rate of ATP gamma S turnover is severalfold (4-8) slower than ATP turnover, the rate-limiting step must be release of thiophosphate, not ADP. Thus, microtubules can activate the rate of thiophosphate release. The stereochemical course of phosphoric residue transfer was determined by using ATP gamma S stereospecifically labeled in the gamma position with 18O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of recA protein. The open helix model for LexA cleavage.

RecA protein is induced by the binding of DNA and ATP to become active in the hydrolysis of ATP and the cleavage of repressors. These reactions appear to depend on the structural state of the protein polymerized along the DNA, i.e. a helical coat of six RecA per turn of 95 to 100 A pitch. In support of this model of the active conformation, it was shown that high concentrations of salt also induce this helical polymerized state as well as the enzymatic activities. Here, we describe that, in vitro and with the non-hydrolyzable analogue ATP gamma S, RNA and heparin can also induce both the structural transition and the enzymatic activation of RecA to LexA cleavage in accordance with the model. RNA and heparin do not support the reaction in the presence of ATP, and they do not induce the hydrolysis of ATP either, suggesting that, in contrast to ATP gamma S, the nucleotide is not bound stably enough, and that the combined affinities of polynucleotide and ATP actually modulate the discrimination of RecA for the various possible inducers in vivo.     

Effects of Mg2+ and the beta gamma-subunit complex on the interactions of guanine nucleotides with G proteins

Mg2+ interacts with the alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) in the presence of guanosine-5'-[gamma-thio]triphosphate (GTP-gamma S) to form a highly fluorescent complex from which nucleotide dissociates very slowly. The apparent Kd for interaction of G alpha X GTP gamma S with Mg2+ is approximately 5 nM, similar to the Km for G protein GTPase activity X G beta gamma increases the rate of dissociation of GTP gamma S from G alpha X GTP gamma S or G alpha X GTP gamma S X Mg2+ at low concentrations of Mg2+. When the concentration of Mg2+ exceeds 1 mM, G beta gamma dissociates from G beta gamma X G alpha X GTP gamma S X Mg2+. Compared with the dramatic effect of Mg2+ on binding of GTP gamma S to G alpha, the metal has relatively little effect on the binding of GDP. However, G beta gamma increases the affinity of G alpha for GDP by more than 100-fold. High concentrations of Mg2+ promote the dissociation of GDP from G beta gamma X G alpha X GDP, apparently without causing subunit dissociation. The steady-state rate of GTP hydrolysis is strictly correlated with the rate of dissociation of GDP from G alpha under all conditions examined. Thus, there are at least two sites for interaction of Mg2+ with G protein-nucleotide complexes. Furthermore, binding of G beta gamma and GTP gamma S to G alpha is negatively cooperative, while the binding interaction between G beta gamma and GDP is strongly positive.     

Kinetic studies of recA protein binding to a fluorescent single-stranded polynucleotide

Fluorescence spectroscopy was used to investigate the binding of Escherichia coli recA protein to a single-stranded polynucleotide. Poly(deoxy-1,N6-ethenoadenylic acid) was prepared by reaction of chloroacetaldehyde with poly(deoxyadenylic acid). The fluorescence of poly(deoxy-1,N6-ethenoadenylic acid) was enhanced upon recA protein binding. The kinetics of the binding process were studied as a function of several parameters: ionic concentration (KCl and MgCl2), pH, nature of the nucleoside triphosphate [adenosine 5'-triphosphate or adenosine 5'-O-(gamma-thiotriphosphate)], protein and polynucleotide concentrations, polynucleotide chain length, and order of sequential additions. The observed kinetic curves exhibited a lag phase followed by a slow binding process characteristic of a nucleation-elongation mechanism with an additional slow step governing the rate of the association process. The lag phase reflecting the nucleation step was not observed when the protein was first bound to the polynucleotide before addition of adenosine 5'-triphosphate. Adenosine 5'-triphosphate induced a dissociation of the recA protein, which was immediately followed by binding of the recA-adenosine 5'-triphosphate-Mg2+ ternary complex. The origin of this "mnemonic effect" and of the different kinetic steps is discussed with respect to protein conformational changes and aggregation phenomena.     

Linear dichroism study of RecA-DNA complexes. Structural evidence and binding stoichiometries

In the presence of RecA single-stranded DNA (ssDNA) is found to exhibit flow linear dichroism (LD). In the absence of the cofactor ATP gamma S, the LD is positive with a maximum at about 280 nm, whereas in the presence of the cofactor ATP gamma S there is still a positive long-wavelength band, but a negative LD contribution centered at 260 nm indicates an orientation of the DNA bases preferentially perpendicular to the fiber axis. For the complex between ssDNA and RecA without ATP gamma S, essentially all LD derives from the protein (tryptophane) subunits indicating a structure in which the tryptophanes are preferentially parallel to the fiber axis of the complex while the DNA bases remain essentially unoriented. The magnitude of the LD increases with the RecA/DNA ratio to a point corresponding to approximately three nucleotides per RecA and decreases thereafter with excess of DNA. This indicates that there are two modes of binding with different stoichiometries.     

Porphyrin and metalloporphyrin binding to DNA polymers: rate and equilibrium binding studies

Interactions of meso-tetrakis(4-N-methylpyridiniumyl)porphyrin [TMpyP(4)] with poly[d(G-C)].poly[d(G-C)] [poly[d(G-C)2] and poly[d(A-T)].poly[d(A-T)] [poly[d(A-T)2] were studied by equilibrium dialysis and stopped-flow dissociation kinetics as a function of [Na+]. Metalloderivatives of TMpyP(4), NiTMpyP(4), and ZnTMpyP(4) were also investigated. The apparent equilibrium binding constants (Kobs) were approximately the same for TMpyP(4) binding to either poly[d(G-C)2] or poly[d(A-T)2] and decreased with increasing [Na+]. The slopes of the plots of log Kobs vs log [Na+] were similar, with values close to -2.7. Contrary to implications in previously reported studies, these data do not indicate that TMpyP(4) prefers to bind to GC sites at low ionic strength and to AT sites at high ionic strength. In contrast, binding of ZnTMpyP(4) to these two polymers is very different. Comparisons of Kobs values at 0.065 M [Na+] indicate that ZnTMpyP(4) binding to AT sites is approximately 200 times more favorable than binding to GC sites, a finding in agreement with previous qualitative observations. Although the binding of the Zn species to the GC polymer was too weak for us to assess the salt effect, the plot of log Kobs vs log [Na+] gave a slope of -2.0 for ZnTMpyP(4) binding to poly[d(A-T)2]. Application of condensation theory for polyelectrolytes suggests similar charge interactions for ZnTMpyP(4) and for TMpyP(4) binding to poly[d(A-T)2]. Likewise, the rates of dissociation from poly[d(A-T)2] were similar for TMpyP(4) and ZnTMpyP(4) [and also NiTMpyP(4)]. However, whereas TMpyP(4) [and NiTMpyP(4)] dissociation from poly[d(G-C)2] was measurable, that for ZnTMpyP(4) was too fast to measure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Function of nucleoside triphosphate and polynucleotide in Escherichia coli recA protein-directed cleavage of phage lambda repressor

Escherichia coli recA protein catalyzes a specific proteolytic cleavage of repressors in vitro when it is activated by interaction with a single-stranded polynucleotide and nucleoside triphosphate. The ATP analogue adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S) satisfies the NTP requirement. We show here that despite its activity in repressor cleavage, ATP gamma S is hydrolyzed at a negligible rate by the recA protein DNA-dependent nucleoside triphosphatase activity. In the presence of DNA, ATP gamma S binds tightly to recA protein in a complex that can be detected because it is trapped by a nitrocellulose filter. One ATP gamma S molecule is bound per recA monomer. These results suggest that a ternary complex of recA protein, DNA, and nucleoside triphosphate is the species active in repressor cleavage. The activation of recA protein by small, defined oligonucleotides in place of DNA is described and characterized.     

Inhibitory effects of ATP and other nucleotides on atrial natriuretic peptide (ANP) binding to R1-type ANP receptors in human neuroblastoma NB-OK-1 cell membranes.

ATP dose-dependently inhibited rat 125I-ANP-(99-126) binding to membranes from the human neuroblastoma cell line NB-OK-1 by increasing the KD value for the hormone without altering the Bmax value. After a 20 min preincubation with 37.5 pM 125I-ANP-(99-126) and 0.5 mM ATP, followed by the addition of 0.3 microM unlabelled ANP-(99-126), the proportion of rapidly dissociating receptors was 4-times higher than in the absence of ATP. The other nucleotides ADP, AMP, AMP-PNP, ATP gamma S, GTP, GDP, GMP, GMP-PNP and GTP gamma S were also inhibitory but with a lower potency and/or efficacy. Binding equilibrium data were satisfactorily simulated by a computer program based on partially competitive binding of ANP-(99-126) and the nucleotides, and this, together with the data on dissociation kinetics, strongly suggests that several nucleotides, when added at concentrations up to 1 mM, form a ternary ANP-receptor-nucleotide complex.     

Initial rate kinetic analysis of the mechanism of initiation complex formation and the role of initiation factor IF-3.

Initial rate kinetics of the formation of ternary complexes of Escherichia coli 30S ribosomal subunits, poly(uridylic acid), and N-acetylphenylalanyl transfer ribonucleic acid in the presence and in the absence of IF-3 are consistent with the hypothesis that the ternary complex is formed through a random order of addition of polynucleotide and aminoacyl-tRNA to separate and independent binding sites on the 30S ribosomes. The transformation of an intermediate into a stable ternary complex which probably entails a rearrangement of the ribosome structure leading to a codon-anticodon interaction represents the rate-limiting step in the formation of the ternary complex. The rate constant of this transformation, as well as the association constants for the formation of the 30S-poly(U) and 30S-N-AcPhe-tRNA binary complexes, are enhanced by the presence of IF-3 which acts as a kinetic effector on reactions which are intrinsic properties of the 30S ribosome. The IF-3-induced modification of these kinetic parameters of the 30S ribosomal subunit can per se explain the effect of IF-3 on protein synthesis without invoking a specific action at the level of the mRNA-ribosome interaction. This seems to be confirmed by the finding that IF-3 can stimulate several-fold the formation of a ternary complex even if one by-passes the ribosome-template binding step by starting with a covalent 30S-polynucleotide binary complex. Furthermore, the above-mentioned changes induced by IF-3 appear to be compatible with the previously proposed idea that the binding of the factor modifies the conformation of the 30S subunit. The random order of addition of substrates determined for the 30S-N-AcPhe-tRNA-poly(U) model system was found to be valid also for the more physiological 30S initiation complex containing poly(A,U.G) and (fMet-tRNA formed at low Mg2+ concentration in the presence of GTP and all three initiation factors.     

Binding stoichiometry and structure of RecA-DNA complexes studied by flow linear dichroism and fluorescence spectroscopy. Evidence for multiple heterogeneous DNA co-ordination

The interaction between RecA and DNA (in the form of unmodified single-stranded DNA, fluorescent single-stranded DNA and double-stranded DNA) is studied with linear dichroism and fluorescence spectroscopy. RecA is found to form a complex with single-stranded DNA with a binding stoichiometry of about four nucleotides per RecA monomer, in which the DNA bases appear to have a random orientation. Addition of ATP gamma S (a non-hydrolyzable analog of ATP) reduces the stoichiometry to about three nucleotides per RecA and causes the DNA bases to adopt an orientation preferentially perpendicular to the fiber axis. This complex can incorporate an additional strand of single-stranded DNA or double-stranded DNA, yielding a total stoichiometry of six nucleotides or three nucleotides and three base-pairs, respectively, per RecA. RecA, in the presence of ATP gamma S, is also found to interact with double-stranded DNA, with a stoichiometry of about three base-pairs per RecA. In all studied complexes, the tryptophan residues in the RecA protein are oriented with their planes preferentially parallel to the fiber axis, whereas in complexes involving ATP gamma S the planes of the DNA bases are oriented preferentially perpendicular to the fiber. This virtually excludes the possibility that the tryptophan residues are intercalated in the DNA helix. On the basis of these results, a model for the research of homology in the RecA-mediated, strand-exchange reaction in the genetic recombination process is proposed.     

ATP-induced dissociation of rabbit skeletal actomyosin subfragment 1. Characterization of an isomerization of the ternary acto-S1-ATP complex

The adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) induced dissociation of actomyosin subfragment 1 (S1) has been investigated by monitoring the light scattering changes that occur on dissociation. We have shown that ATP gamma S dissociates acto-S1 by a mechanism similar to that of ATP but at a rate 10 times slower. The maximum rate of dissociation is limited by an isomerization of the ternary actin-S1-nucleotide complex, which has a rate of 500 s-1 for ATP gamma S and an estimated rate of 5000 s-1 for ATP (20 degrees C, 0.1 M KCl, pH 7.0). The activation energy for the isomerization is the same for ATP and ATP gamma S, and both show a break in the Arrhenius plot at 5 degrees C. The reaction between acto-S1 and ATP was also followed by the fluorescence of a pyrene group covalently attached to Cys-374. We show that the fluorescence of the pyrene group reports the isomerization step and not actin dissociation. The characterization of this isomerization is discussed in relation to force-generating models of the actomyosin cross-bridge cycle.     

N-terminal deletion of the gamma subunit affects the stabilization and activity of chloroplast ATP synthase

Five truncation mutants of chloroplast ATP synthase gamma subunit from spinach (Spinacia oleracea) lacking 8, 12, 16, 20 or 60 N-terminal amino acids were generated by PCR by a mutagenesis method. The recombinant gamma genes were overexpressed in Escherichia coli and assembled with alphabeta subunits into a native complex. The wild-type (WT) alphabetagamma assembly i.e. alphabetagammaWT exhibited high (Mg2+)-dependent and (Ca2+)-dependent ATP hydrolytic activity. Deletions of eight residues of the gamma subunit N-terminus caused a decrease in rates of ATP hydrolysis to 30% of that of the alphabetaWT assembly. Furthermore, only approximately 6% of ATP hydrolytic activity was retained with the sequential deletions of gamma subunit up to 20 residues compared with the activity of the alphabetaWT assembly. The inhibitory effect of the epsilon subunit on ATP hydrolysis of these alphabetagamma assemblies varied to a large extent. These observations indicate that the N-terminus of the gamma subunit is very important, together with other regions of the gamma subunit, in stabilization of the enzyme complex or during cooperative catalysis. In addition, the in vitro binding assay showed that the gamma subunit N-terminus is not a crucial region in binding of the epsilon subunit.     

Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system

Day, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system. J. Bacteriol. 91:1917-1923. 1966.-Tetracycline, an inhibitor of cell-free protein synthesis, effected the dissociation of Escherichia coli 100S ribosomes to 70S particles in vivo and in vitro, but was not observed to mediate the further degradation of these particles. The antibiotic was bound by both 50S (Svedberg) and 30S subunits of 70S ribosomes and also by E. coli soluble RNA (sRNA), polyuridylic acid (poly U), and polyadenylic acid (poly A). The binding to ribosomal subunits was higher at 5 x 10(-4)m Mg(++) than at 10(-2)m Mg(++). The binding to polynucleotide chains was highest when Mg(++) was not added to the reaction mixture.     

Inhibition of recA protein promoted ATP hydrolysis. 1. ATP gamma S and ADP are antagonistic inhibitors

ADP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) inhibit recA protein promoted ATP hydrolysis by fundamentally different mechanisms. In both cases, at least two modes of inhibition are observed. For ADP, the first mode is competitive inhibition. The second mode is manifested by dissociation of recA protein from DNA. These are readily distinguished in a comparison of ATP hydrolyses that are activated by (a) DNA and (b) high (approximately 2 M) salt concentrations. Competitive inhibition with a significant degree of cooperativity is observed under both sets of conditions, although the DNA-dependent activity is more sensitive to ADP than the high-salt reaction. The reaction in the presence of poly(deoxythymidylic acid) or duplex DNA ceases when about 60% of the available ATP is hydrolyzed, reflecting an ADP-mediated dissociation of recA protein from the DNA that is governed by the ADP/ATP ratio. In contrast, ATP hydrolysis proceeds nearly to completion at high salt concentrations. At high concentrations of ATP and ATP gamma S, ATP gamma S also acts as a competitive inhibitor. At low concentrations of ATP gamma S and ATP, however, ATP gamma S activates ATP hydrolysis. These patterns are observed for recA-mediated ATP hydrolysis with either high salt concentrations or a poly(deoxythymidylic acid) [poly(dT)] cofactor, although the activation is observed at much lower ATP and ATP gamma S concentrations when poly(dT) is used. ATP gamma S can also relieve the inhibitory effect of ADP under some conditions. ATP gamma S and ADP are antagonistic inhibitors, reinforcing the idea that they stabilize different conformations of the protein and suggesting that these conformations are mutually exclusive. The ATP gamma S (ATP) conformation is active in ATP hydrolysis. The ADP conformation is inactive.