Nanosecond Fluorescence Depolarization Research Articles

Molecular structural studies of effector functions of a mouse immunoglobulin G that lacks the entire C H1 domain: Small-angle X-ray scattering, nanosecond fluorescence depolarization and stable isotope-aided NMR analyses

Molecular structural studies are reported of a short-chain mouse IgG2a antibody that lacks the entire C H1 domain. We have recently shown that (1) this short-chain antibody comprises two components in which the inter light-chain disulflde bridge does and does not exist, and (2) these two components are different in the constitutive complement-activating activity [Mizutani et al. (1993) J. Immunol. 150, 131–138]. Structures were compared for these two components on the basis of small-angle X-ray scattering, nanosecond fluorescence depolarization and isotope-aided NMR data. It has been discussed how the presence and absence of the inter light-chain disulfide bridges affect the complement-activating activity of the two components of the short-chain antibody.

A genetically engineered human IgG with limited flexibility fully initiates cytolysis via complement

A causal link between antigen induced conformational change and functional activity has been previously suggested for the triggering of immunoglobulin effector functions. To test this hypothesis an immunoglobulin with greatly restricted Fab flexibility has been created by site-directed mutagenesis. Serine 119, a residue at the elbow in the first heavy chain constant domain of a mouse-human chimeric immunoglobulin, was replaced by cysteine 119. The resulting mutant immunoglobulin had an additional intramolecular disulfide bond connecting the two heavy chains. This newly introduced covalent bond between antigen binding arms gave rise to a compact ‘tethered’ conformation which displayed lowered segmental flexibility as determined by nanosecond fluorescence depolarization anisotropy. However, the ability of this tethered immunoglobulin to initiate lysis of target cells via the complement cascade was unimpaired. Therefore, it is likely that allosteric or distortive mechanisms of conformational change induced complement activation which require unhindered ‘twist’ and/or ‘waggle’ motions of Fab are untenable.

Changes of fluorescence lifetime and rotational correlation time of bovine serum albumin labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride in guanidine and thermal denaturations

The nanosecond fluorescence depolarization method was applied to measure the fluorescence lifetime (tau) and the rotational correlation time (phi) of bovine serum albumin (BSA) labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-Cl). Changes of tau and phi of dansyl BSA in the guanidine denaturation and in the thermal denaturation were examined. In parallel, the secondary structural change of dansyl BSA was followed by circular dichroism measurements. The magnitude of tau was almost unchanged between 1 and 2M guanidine, where the secondary structure of the protein was predominantly disrupted; whereas that of phi began to increase before the disruption of secondary structure in the guanidine denaturation. In the thermal denaturation, in contrast, changes of both tau and phi occurred in a temperature range where the secondary structure was predominantly disrupted. The volume of equivalent sphere (Ve) and the axial ratio (rho) for the BSA were 3.6-3.8 x 10(-19) cm3 and 3.6 at 2 M guanidine as against 2.1 x 10(-19) cm3 and 2.2 in the absence of guanidine (25 degrees C), respectively. The magnitudes of Ve and rho were 4.9 x 10(-19) cm3 and 4.5 at 65 degrees C, respectively. Although the secondary structural change of dansyl BSA was irreversible in the thermal denaturation, Ve and rho were reversible.

A nanosecond fluorescence depolarization study on the segmental flexibility of receptor-bound immunoglobulin E

Time-resolved fluorescence anisotropy measurements have been used to examine the segmental flexibility of anti-dansyl immunoglobulin E (IgE) bound to its high-affinity receptor on membrane vesicles from rat basophilic leukemia cells. Although IgE in this complex exhibits only a restricted angular range of segmental motion, much of this restricted motion occurs on a relatively rapid time scale. A fast component of motion with a rotational correlation time of 15-35 ns may correspond to the twisting of Fab segments about their major axis. Intermolecular cross-linking by a short bivalent ligand, N,N'-didansylcadaverine, results in complete loss of this segmental motion. Solubilization of monomeric IgE-receptor complexes using a zwitterionic detergent results in a time-dependent anisotropy decay that exhibits both a fast component and a slower component that is intermediate between the decay for soluble and membrane-bound forms of IgE at long times after excitation. These results are discussed in terms of a model in which binding of IgE to its membrane-bound receptor restricts not only its global rotation but also its slower modes of segmental flexibility as well, while allowing its Fab segments to undergo rapid reorientation within a limited angular range.

Fluorescence lifetime and rotational correlation time of bovine serum albumin-sodium dodecyl sulfate complex labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride: Effect of disulfide bridges in the protein on these fluorescence parameters

A fluorescent dye, 1-dimethylaminonaphthalene-5-sulfonyl chloride, was used to label bovine serum albumin (BSA), intact and disulfide bridges-cleaved. The fluorescence lifetime and fluorescence anisotropy of the adducts in sodium dodecyl sulfate (SDS) solutions were studied by the nanosecond fluorescence depolarization method. The volume of equivalent sphere (Ve) was calculated to be 2.1 x 10(-19) cm3 for BSA with the intact disulfide bridges from the rotational correlation time. The value of Ve was 4.4 x 10(-19) cm3 for the disulfide bridges-cleaved BSA. With an increase in SDS concentration, the rotational correlation time of the intact BSA became longer, while that of the disulfide bridges-cleaved BSA became shorter. This suggests that upon the binding of SDS, the total volume of the intact BSA increases while the expanded state of the protein, caused by the cleavage of the disulfide bridges, becomes compact.

A fluorescence photobleaching study of the microsecond reorientational motions of DNA

We have conducted a polarized fluorescence photobleaching recovery (FPR) study of the rotational dynamics of ethidium azide labeled DNA. Polarized photobleaching experiments provide data on microsecond and millisecond molecular reorientation that complement the information available from nanosecond fluorescence depolarization studies. In polarized FPR experiments an anisotropic angular concentration of fluorophore is created by bleaching dye molecules in a preferred orientation with a short, intense pulse of polarized light. The sample is then weakly illuminated, and the temporal variation in the emitted fluorescence is monitored. The fluorescence signal will systematically change as molecules undergo post-bleach reorientation and the angular distribution of dye tends toward isotropy. We have observed that the time dependence of our microsecond FPR curves is also determined in part by nonrotational phenomena. To isolate the reorientational recovery we conduct our FPR experiments in two modes (called parallel and perpendicular) that differ only in the polarization of the bleaching light. A quotient function, R(t), is constructed from the data obtained in these two modes; the variation with time of this new quantity is governed solely by processes that are sensitive to the polarization of the incident light (e.g., molecular rotation). It is found experimentally that R(t) remains constant, as expected, for rotationally restricted DNA systems despite a temporal recovery in the parallel and perpendicular FPR curves. We also follow the dynamics of solutions of phage lambda DNA as revealed in the temporal dependence of R(t). This DNA system rotationally relaxes after approximately 100 microseconds and the dye/DNA complex reorients substantially during the 10-microseconds bleach period. Our FPR data are interpreted in terms of dynamic models of DNA motion.

Oxygen diffusion coefficient of cell membranes.

The microviscosity was measured in erythrocyte membranes of different animal species and in lung cells and myocytes of bull frogs by means of the nanosecond fluorescence depolarization technique with a rod-like fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). The diffusion coefficient of oxygen molecules (DO2) was estimated from the microviscosity and an equation for the relation between viscosity and diffusion coefficient. The DO2 value of erythrocyte ghosts was remarkably different among species; 0.84 in sheep, 1.29 in human, 1.49 in rat and 1.92 X 10(-7) cm2/sec in rabbit, while the wobbling diameter Wd was 10.7, 11.5, 12.1 and 10.7A in respective species. The DO2 value in frogs was different among organs; 1.07 in erythrocyte ghosts, 2.18 in lung cells, 2.64 X 10(-7) cm2/sec in myocytes. The Wd value was 12.2, 12.6 and 13.4A in respective organs at 30 C. A comparison between sizes of moving area of phospholipid molecules in membrane and in ideal fluid state suggested that the actual DO2 values in cell membranes may probably be ten times larger than the present values.

Some theoretical considerations regarding the effects of steric hindrance and intrinsic global coupling on the flexibility of Fc-anchored immunoglobulins

Nanosecond fluorescence depolarization studies reported in the accompanying companion paper showed that the long rotational correlation time, φ L , increased somewhat when rabbit IgG anti-dansyl antibodies were anchored in staphylococcal protein A (SpA) soluble complexes. The increases in φ L upon anchoring IgG probably resulted from “global coupling” effects caused by: (1) increased steric hindrance of the antibody segments in the SpA complexes and (2) intrinsic structural constraints already present in the monomeric IgG. Global coupling results from a restriction in the angular range of a flexible segment and is manifest when flexible motions alone cannot depolarize all of the fluorescence, so that the slower global tumbling of the entire particle is also required. Such effects cannot be resolved directly from experimental anisotropy data, however, because only a single long correlation time, φ L , is well defined over the limited time range of most fluorophores. In this paper, estimates of the anisotropy contributions from flexible and global motions of the IgG-SpA complexes are determined by contrasting theoretical and measured decays. For this analysis it was assumed that each of the experimental φ L -values is a weighted composite of the rotational correlation time associated with the less restricted flexible motions of the Fab arms, φ F , and the correlation time associated with global tumbling of the entire particle, φ G . A general two-exponential expression was used to relate φ F and φ G to φ L . This approach was meaningful because φ G -values of the various SpA complexes had been calculated from hydrodynamic measurements. The theoretical decays clearly show that, even if φ G is much longer than φ F , these two rotational motions still cannot be resolved over the experimentally accessible time range. Families of emission anisotropy decay curves for IgG antibodies with different amounts of intrinsic global coupling and for anchored antibodies with different amounts of steric hindrance were simulated by varying the preexponential weighting factors of the flexible and global terms. By comparing the calculated curves with the measured decays, it is evident that the rabbit IgG anti-dansyl antibodies do not have much intrinsic global coupling, but rather they are highly flexible. The curves also indicate that even for the exceptionally compact IgG 4-SpA 2 17-S complex, which showed the most steric hindrance in electron micrographs, the appropriate φ G weighting factor is only 0.28. Thus, as supposed earlier, the anchored antibodies exhibit considerable segmental flexibility. In closing, the above concepts are used to examine the results of other reported depolarization studies with mouse monoclonal IgG1 antibodies.

Internal motion of deoxyribonucleic acid in chromatin. Nanosecond fluorescence studies of intercalated ethidium.

We have investigated the internal motions of DNA in a nucleosome core particle and chromatin by measuring the nanosecond fluorescence depolarization of intercalated ethidium. Assuming that the observed anisotrophy decay originates from the torsional motion of DNA, we have analyzed the dynamics of DNA in a nucleosome core particle and in chromatin in detail. The results suggest that DNA in a nucleosome core particle has a torsional rigidity similar to that of DNA in solution and that even at the point of the ionic bonds between DNA and a histone octamer the torsional motion of DNA is not completely inhibited. On the other hand, the dynamics of linker DNA in chromatin were found to reflect the overall structural state of the chromatin: the motion of linker DNA was suppressed as the structure of chromatin turned from an extended state to a condensed one. This indicates that, in solenoidal chromatin, nucleosome movements in chromatin are largely suppressed. Furthermore, the result may suggest that the torsional rigidity of linker DNA is increased as it is forced to bend in solenoidal chromatin.

The effect of cytochrome oxidase on lipid chain dynamics A nanosecond fluorescence depolarization study

Molecular motions in membranes composed of purified cytochrome oxidase (EC 1.9.3.1) and synthetic lipid ( l-α-dimyristoylphosphatidylcholine or l-α-dioleoylphosphatidylcholine) at various ratios were investigated with a lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Nanosecond fluorescence depolarization kinetics of the probe showed that the rod-shaped probe molecules perform a fast wobbling motion (restricted rotation) in all membranes studied, presumably reflecting the motion of lipid acyl chains. At temperatures where the pure lipid was in the liquid-crystalline phase, presence of cytochrome oxidase reduced the angular range of the wobbling motion, whereas its rate; the wobbling diffusion constant, was unaffected. On the other hand, incorporation of the protein into lipid in the gel phase resulted in the increase in the wobbling diffusion constant while the range of the wobbling motion remained the same. A time-dependent view of lipid dynamics that accounts for the above findings, as well as the results of recent electron spin resonance and nuclear spin resonance studies of protein-lipid interactions, is proposed.

Dynamic structure of biological membranes as probed by 1,6-diphenyl-1,3,5-hexatriene: a nanosecond fluorescence depolarization study.

A fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene, was incorporated in four different biological membranes, the purple membrane of Halobacterium halobium, human erythrocyte membrane, rabbit sarcoplasmic reticulum membrane, and rat liver mitochondrial membrane. Time-resolved fluorescence depolarization of the probe suggested that the rotational Brownian motion of the probe in the membranes was restricted in the angular range. The motion of the rod-shaped, lipophilic probe molecule, expected to reflect closely the motion of neighboring lipid hydrocarbon chains, was analyzed in terms of the wobbling-in-cone model in which the major axis of the probe was assumed to wobble freely in a cone of semiangle theta c with a wobbling diffusion constant Dw. At 35 degrees C, Dw in the four membranes, in the above order, ranged between 0.048 and 0.15 ns-1 and theta c between 31 and 53 degrees. From the rotational rate Dw, the viscosity against the wobbling motion was calculated to be 0.9-0.3 P. When the temperature was raised from 10 to 35 degrees C, Dw in all membranes increased approximately 3-fold, corresponding to activation energies of 7-8 kcal/mol, and theta c increased by about 10 degrees, except for the purple membrane in which the angular range remained narrow. The same characteristic temperature dependence has been found in many model membrane systems that contain unsaturated lecithins, suggesting an important role of unsaturated phospholipids in the dynamic structure of the lipid hydrocarbon chain region of biological membranes at physiological temperatures. Comparison with model systems suggests that proteins and cholesterol act mainly as barriers that narrow the angular range.

Local mobility of the lac repressor molecule

The rotational mobility of lac repressor from Escherichia coli was investigated by nanosecond fluorescence depolarization spectroscopy. A single rotational correlation time (φ) of the repressor was observed by monitoring the emission anisotropic decay of the intrinsic tryptophan fluorescence. The small value of φ (9·5 ns) suggests that one or both of the two tryptophan residues in the repressor are located in a flexible segment of the protein molecule. This segmental flexibility is enhanced by binding of inducer (isopropyl-β- d-thiogalactoside) to the repressor while it is restrained by binding of anti-inducer (glucose) or small DNA fragments, as indicated by the changes in φ. Further time-dependent emission anisotropy studies with an extrinsic fluorescent probe, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate, covalently attached to the repressor yielded two rotational correlation times. The shorter φ S (6·7 ns) also corresponds to a segmental flexibility whereas the longer φ L (118 ns) represents the rotational motion of the entire repressor molecule. Both the values of φ S and φ L vary by addition of inducer or anti-inducer in a manner similar to that observed for the intrinsic tryptophan fluorescence but they are insensitive to addition of DNA fragments. The changes in local mobility of the lac repressor molecule observed in these studies may provide some insight into how inducer (or anti-inducer) destabilizes (or stabilizes) the repressor-operator complex.

Intramolecular excimer formation in polyamides with widely separated naphthyl groups in the backbone

AbstractThe photophysical properties of a series of polyamides containing naphthalene groups in the backbone were investigated. The polyamides were synthesized by interfacial polycondensation involving 2,6‐bis(N‐methyl, methylamino)naphthalene and the following diacid chlorides: magnified image (x = 2, 4, 6, and 8). The polyers were designated P‐2, P‐4, P‐6, and P‐8. The monomer model compound (MC) was made by acylation of the diamine monomer wth acetyl chloride. The polyamides exhibited intramoleular excimer fluorescence in dilute solutions at room temperature. Excimer fluorescence quantum yields were in the following order: P‐2 ≈ P‐8 > P‐4 ≈ P‐6. This sequence was rationalized on the basis of neighboring and nonneighboring group excimeric interactions that are dependent on the length of the intervening methylee chain. Temperature measurements on P‐2 revealed a small energy of actvation (1.8 kcal mole−1) for the conformational transition involved in the formaton of the intramolecular excimer. Intramolecular excimer formation for a given polymer depended on the solvent. Nanosecond fluorescence depolarization measurements demonstrated the flexibility of the naphthalene moieties even though they were doubly bound in the polymer chain. Our results showed that intramolecular excimer formation could occur over long distances of separation (n > 3) between the nteracting chromophores provided that the connecting chain was sufficiently flexible.

Interaction of .alpha.-chymotrypsin with the fluorescent probe 1-anilinonaphthalene-8-sulfonate in solution

The binding of the fluorescence probe 1-anilinonaphthalene-8-sulfonate (Ans) to alpha-chymotrypsin (alpha-CHT) at pH 3.6 is accompanied by a dramatic enhancement of Ans fluorescence and a shift of the emission maximum to shorter wavelengths. Our study reveals that one Ans molecule binds to alpha-CHT at a site different from either the active site of alpha-CHT or the 2-p-toluidinylnapthalene-6-sulfonate binding site. the binding constant of Ans is about the same (10(4) M-1) at pH 3.6 and 6.4. Nanosecond fluorescence depolarization data indicate that Ans is rigidly bound to alpha-CHT. The fluorescence enhancement due to binding of Ans to alpha-CHT at low pH could be due to binding either to a hydrophobic site or to a site where local dipoles do not relax during the excited-state lifetime of Ans. As the pH is increased, fluorescence intensity of the Ans-alpha-CHT complex decreases appreciably; and the emission maximum shifts to longer wavelengths. The fluorescence decay curves exhibit a corresponding sensitivity to pH. The pH effect on the fluorescence of Ans-alpha-CHT can be interpreted in terms of a pH-dependent equilibrium between alpha-CHT conformers differing in the degree of mobility of polar residues and water molecules at the Ans binding site or structural changes in the Ans binding site.

Theory of twisting and bending of chain macromolecules; analysis of the fluorescence depolarization of DNA

An elastic model of semiflexible chain macromolecules is developed in order to treat internal rotatory Brownian motion in the DNA helix. Dynamical equations for torsion and bending of the chain are generated, using results from classical elasticity and hydrodynamic theories. The rotational diffusion equation in normal coordinates is derived, and the initial-boundary value problem solved for the conditions of a nanosecond fluorescence depolarization experiment. The resulting time distribution function of the angular orientation of a fluorescent probe, embedded in a chain at thermal equilibrium, is used to compute the emission anisotropy. The predicted decay law is unusual, with exponentials in ∼t due to twisting and in ∼t1/4 due to bending. Comparison with published data for ethidium–DNA complex reveals that the decay of the anisotropy arises primarily from twisting of the DNA helix, with a small contribution from bending. By fitting theory and experiment, the torsional rigidity C of DNA may be obtained.

Fibrinogen: Agreement of Experimental and Calculated Hydrodynamic Data with Electron-Microscopic Models

There is no general agreement on the size and shape of the fibrinogen molecule. We have studied the diffusion of the fibrinogen in solution by means of dynamic light scattering, nanosecond fluorescence depolarization and analytical ultracentrifugation. The results obtained under physiological concentration, pH an ionic strength are DT = 2.0 X 10(7) cm2sec-1, DR perpendicular = 40'000 sec 1. Nanosecond fluorescence depolarization yielded DR parallel = 1.6 x 10(6) sec-1. Tentatively this value is interpreted as DR parallel, namely rotational diffusion about the major axis of the molecule. The sedimentation coefficient is 8.1 S. The hydrodynamic parameters derived from our measurements were compared with those calculated on the basis of the models proposed by Hall and Slayter, Hudry-Clergeon and Marguerie et al., Bachmann and Lederer, and Köppel. The agreement is poor even if the degree of hydration is varied within wide limits. However, satisfactory agreement can be achieved by assuming a flexible molecule of about 900 A length corresponding to two end-to-end bound trinodular structures of the Hall and Slayter type, with a nodule diameter of about 45 A. Experimental evidence indicates that the discrepancies between the different models might be due to different techniques of sample preparation leading to different conformations of the molecule.

Nanosecond fluorescence depolarization studies on actin labeled with 1,5-IAEDANS and dansyl chloride: Evidence for label flexibility

Nanosecond fluorescence depolarization studies on actin labeled with 1,5-IAEDANS and dansyl chloride: Evidence for label flexibility

Nanosecond fluorescence spectroscopy of pyrenebutyrate anti-pyrene antibody complexes.

The utility of the long-lived fluorophore, pyrene, as a probe in nanosecond fluorescence depolarization measurements was investigated using pyrenebutyrate bound in the combining sites of rabbit antipyrenebutyrate immunoglobulin G. The time dependent anisotropy decay data points showed very little scatter in the time interval 0-350 ns, which is more than three times the comparable time interval observed with epsilon-1-dimethylamino-5-naphthalenesulfonyllysine (DNS-lysine) bound in the combining sites of anti-DNS antibodies [Holowka, D.A., and Cathou, R.E. (1976), Biochemistry 15, 3379]. Thus, the use of pyrene can significantly extend the range of macromolecular rotational correlation times that can be measured by the single photon technique. In the present investigation, we confirmed the presence of Fab segmental fexibility in immunoglobulin G molecules specific for a hapten different from DNS-lysine. We obtained a value of about 135 ns for the longer rotational correlation time which probably represents global rotation of the entire molecule. In the course of these experiments, we have also found that the combining sites of antipyrenebutyrate antibodies are, as expected, relatively nonpolar.

Spatial relationship of the sigma subunit and the rifampicin binding site in RNA polymerase of Escherichia coli.

sigma subunit of Escherichia coli RNA polymerase is known to stimulate specific RNA chain initiation. Rifampicin, an inhibitor of RNA chain initiation, binds to a single site on the beta subunit of RNA polymerase. We have used the fluorescence energy transfer technique to deduce proximity relationships of sigma subunit and rifampicin binding site on the enzyme. Isolated sigma subunit was covalently labeled with fluorescent donors in two ways: specific labeling of a single sulfhydryl residue with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate (1,5-I-AENS) and nonspecific labeling on the surface of the protein with dansyl chloride (Dns-Cl) adsorbed on Celite. The labeled sigma subunits were biologically active and formed a stoichiometric complex with core polymerase. The efficiency of energy transfer was obtained from the fluorescence intensity and the excited-state lifetime of the sigma-labeled holoenzyme in the presence and absence of rifampicin, which served as an energy acceptor. The transfer efficiency (2%) from AENS to rifampicin placed AENS somewhere between 42 and 85 A away from the rifampicin binding site. The rotational mobility of the donor was determined by nanosecond fluorescence depolarization spectroscopy, while the acceptor orientation was assumed to be fixed at some unknown angle. The efficiency measured for energy transfer from Dns to rifampicin was 10% in the presence of 0.2 M KCl. The distance from the surface of sigma subunit to the rifampicin binding site was calculated to be 27--38 A for a model having a randomly distributed and oriented array of donors on the surface of a spherical sigma subunit of 31-A radius. Our results indicate that rifampicin does not inhibit the initiation of transcription by RNA polymerase through a direct interaction with sigma subunit. In addition, energy transfer measurements under low salt conditions suggest that in RNA polymerase dimer the two rifampicin binding sites are symmetric with respect to each sigma subunit.

Sigma cycle during in vitro transcription: demonstration by nanosecond fluorescence depolarization spectroscopy.

Studies of RNA chain initiation have suggested that the sigma subunit of Escherichia coli RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate: RNA nucleotidyltransferase; EC 2.7.7.6) is released from the enzyme-template complex during transcription and may be reused by another core polymerase. Nanosecond fluorescence depolarization spectroscopy was used to follow the sigma cycle. Isolated sigma subunit labeled with the fluorescent probe dansyl (DNS) chloride bound stoichiometrically to core polymerase and stimulated transcription of phage T7 DNA to the same extent as did unlabeled sigma. DNS-sigma showed an exponential fluorescence anisotropy decay corresponding to a rotational correlation time of about 100 nsec. This value was unaffected by addition of T7 DNA, but increased about 6-fold when core polymerase was added, and increased further when T7 DNA was added. Such increases are expected for the formation of molecular complexes. Using the anisotropy decays for free DNS-sigma and DNS-sigma-core enzyme bound to T7 DNA, we calculated theoretical decay curves for various mixtures of free and bound sigma. Comparison of the observed anisotropy decay with the calculated curves indicated that about 55% of DNA-sigma was released from the enzyme-T7 DNA complex in the presence of four nucleoside triphosphates under low salt conditions. Sigma release did not occur if rifampicin was added prior to addition of four nucleoside triphosphates or if only three nucleoside triphosphates were present. After sigma was released, addition of core polymerase with rifampicin reduced the free sigma to less than 15%, indicating that the released sigma was accessible to the added core enzyme. Thus these studies have provided physical evidence for the sigma cycle during in vitro transcription.