A statistical method is presented for the interpretation of intramolecular distance measurements by the fluorescence energy transfer technique in systems for which the detailed geometries of the donor-acceptor pairs are unknown. This method enables calculation of the probability that a specified distance range corresponds to the actual distance to be measured. It makes use of the numerically calculated probability density function for the distance of interest. The two general systems considered are the single donor-acceptor pair and the multi-donor-single-acceptor transfer. In both systems, the statistical method incorporates the uncertainty in the orientation of the donor and acceptor dipoles. In addition, it can take into account the rotational mobility of the donor dipoles determined by time-dependent emission anisotropy measurements. When more than one donor is involved in the transfer process, the uncertainties associated with the number and location of individual donors and the size and shape of the donor distribution are also incorporated in calculating the distance ranges. Application of the method was demonstrated for a wide range of transfer efficiency and Ro values for the single donor-acceptor system. Specific examples are also presented for interpretation of both single donor-acceptor and multi-donor-single-acceptor energy transfer measurements performed in order to reveal the spatial relationship of the sigma subunit and the rifampicin binding site in the Escherichia coli RNA polymerase (see Wu, C.-W., Yarbrough, L. R., Wu, F. Y.-H., and Hillel, Z. (1976), Biochemistry, preceding paper in this issue). Analysis of these energy transfer data by methods which use average values of the unknown geometrical parameters of the system yielded results similar to those obtained by the statistical method. However, the statistical method represents a more realistic approach to the interpretation of energy transfer measurements since it provides information concerning the entire range of possible distances and their relative likelihood.

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The quaternary structures of Escherichia coli DNA-dependent RNA polymerase holenzyme (alpha 2 beta beta' sigma) and core enzyme (alpha 2 beta beta') have been investigated by chemical cross-linking with a cleavable bifunctional reagent, methyl 4-mercaptobutyrimidate, and noncleavable reagents, dimethyl suberimidate and N,N'-(1,4-phenylene)bismaleimide. A model of the subunit organization deduced from cross-linked subunit neighbors identified by dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the large beta and beta' subunits constitute the backbone of both core and holoenzyme, while sigma and two alpha subunits interact with this structure along the contact domain of beta and beta' subunits. In holoenzyme, sigma subunit is in the vicinity of at least one alpha subunit. The two alpha subunits are close to each other in holoenzyme, core enzyme, and the isolated alpha 2 beta complex. Cross-linking of the "premature" core and holoenzyme intermediates in the in vitro reconstitution of active enzyme from isolated subunits suggests that these species are composed of subunit complexes of molecular weight lower than that of native core and holoenzyme, respectively. The structural information obtained for RNA polymerase and its subcomplexes has important implications for the enzyme-promoter recognition as well as the mechanism of subunit assembly of the enzyme.

present. After a was released, addition of core polymerase with rifampicin reduced the free a to less than 15%, indicating that the released a was accessible to the added core enzyme. Thus these studies have provided physical evidence for the a cycle during in vitro transcription. The transcription of a bacterial genome is primarily mediated by a single RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6). The purified Escherichia coli RNA polymerase holoenzyme has a subunit structure of a2If'a, with molecular weights for the subunits of 40,000, 155,000, 165,000, and 90,000, respectively (1, 2). The holoenzyme can be reversibly dissociated to yield the core enzyme (a2o') and the a subunit. Like the holoenzyme, core polymerase is catalytically active, but differs in transcribing native double-stranded DNA nonspecifically and inefficiently (3, 4).The a subunit by itself has no catalytic function. Its role in gene transcription is 2-fold. (a) The a specificity: a promotes specific initiation of RNA chains that yields asymmetric transcription resembling in vvo RNA products (1). (b) The a cycle: a stimulates RNA synthesis by increasing the rate of initiation through its catalytic reuse by core polymerase (5).Based on their in tdtro transcriptional studies, Travers and Burgess (5) first proposed the following a cycle: a initially forms a complex with core polymerase, which is able to bind to promotor sites on DNA and to initiate specific RNA synthesis. During or after initiation, a is released from the enzyme-DNA complex and may then be reused by another core polymerase molecule to initiate a new RNA chain.Evidence for the physical separation of a from the enzyme-DNA complex after RNA chain initiation has been provided by use of polyacrylamide gel electrophoresis for Azotobacter and E. coli RNA polymerases (6, 7), or by use of sucrose gradient centrifugation for Pseudomonas RNA polymerase (8). However, the evidence is not conclusive because electrical or centrifugal force used could influence the interaction between a and core polymerase. Even if the effect of the external forces is negligible, these studies only indicate a weakening of the a-core polymerase interaction during transcription, resulting in the subsequent separation of a from core polymerase by the electric field or centrifugal force, and do not prove the physical release of a due to RNA synthesis.This communication presents a direct demonstration by nanosecond fluorescence depolarization spectroscopy of the physical release of a during transcription. This technique measures the rate of molecular rotation (Brownian rotational diffusion) and thereby provides information concerning the sizes, shapes, and aggregation states of macromolecules (9, 10) MATERIALS AND METHODS RNA polymerase holoenzyme was purified from cells of E. coli B as described previously (11). The purity of the enzyme was >95% as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The a subunit and core polymerase were...

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.