In the first part of this paper we present a thermodynamic analysis of the elongation phase of transcription in Escherichia coli. The stability of the elongation complex is described by a "free energy of formation" function (delta G zero f) that is a sum of terms for forming (i) a locally denatured 17-base-pair DNA "bubble"; (ii) a constant-length hybrid between the 3'-terminal 12-nucleotide residues of the RNA transcript and the corresponding region of the DNA template strand; and (iii) a set of binding interactions between the polymerase and certain DNA and RNA residues within and near the "transcription bubble". The transcriptional elongation complex is very stable at most positions along a natural DNA template and moves in a highly processive fashion. At these positions, the delta G zero f function provides a quantitative measure of the stability of the elongation complex. Besides allowing for the polymerization of the RNA transcript, the elongation complex also serves to define the context within which transcript termination occurs. In the second part of the paper the thermodynamic analysis is extended to discriminate between template positions at which the elongation complex is stable and positions at which it is rendered relatively unstable by the presence of a string of rU residues at the 3'-terminus of the RNA together with the formation of a specific RNA hairpin just upstream of this point. Most factor-independent (intrinsic) termination events are thermodynamically disallowed at the former positions and are thermodynamically allowed at the latter positions. The extended form of the analysis closely predicts the exact sites of termination at a number of intrinsic terminators (and attenuators) in the E. coli genome. It also correctly predicts bidirectional function for a number of bidirectional terminators. In some cases it may identify terminators that are similar to the intrinsic type but that require additional protein factors, unusual polymerase-nucleic acid interactions, or rate-limiting conformational changes in order to function. Finally, it successfully locates intrinsic terminators within a number of E. coli operons and discriminates between these terminators and the surrounding DNA sequence.

We show that the amino acid analogue betaine shares with small tetraalkylammonium ions [Melchior, W. B., Jr., & von Hippel, P. H. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 298-302] the ability to reduce or even eliminate the base pair composition dependence of DNA thermal melting transitions. The "isostabilizing" concentration of betaine (at which AT and GC base pairs are equally stable) is approximately 5.2 M. Betaine exerts its isostabilizing effect without appreciably altering the conformation of double-stranded DNA from the B form. The presence of > 5 M betaine also does not greatly change the behavior of DNA as a polyelectrolyte; this lack of effect on electrostatic interactions is expected because betaine exists as a zwitterion near neutral pH. Study of DNA melting transitions in high concentrations of betaine thus allows the experimental separation of compositional and polyelectrolyte effects on DNA melting. As a consequence, betaine solutions can also be used to investigate DNA-protein interactions under isostabilizing (or close to isostabilizing) conditions, which has not been possible using isostabilizing salts. This potential is illustrated by examining the highly salt concentration-dependent interaction of ribonuclease A with DNA in concentrated betaine solutions.

In this paper, we develop a kinetic approach to predict the efficiency of termination at intrinsic (factor independent) terminators of Escherichia coli and related organisms. In general, our predictions agree well with experimental results. Our analysis also suggests that termination efficiency can readily be modulated by protein factors and environmental variables that shift the kinetic competition toward either elongation or termination. A quantitative framework for the consideration of such regulatory effects is developed and the strengths and limitations of the approach are discussed. Termination can be experimentally detected on a gel by the appearance of an RNA band of length I that cannot be "chased" into a longer product. Terminators that do not require additional protein factors have been called "intrinsic" (1). Within the limits of experimental resolution (currently -1 sec), termination at an intrinsic terminator appears to occur in an "all-or-none" fashion (2). However, some evidence for a rate-limiting elementary step within the RNA release reaction has been obtained (3); the apparent rate constant for the overall process (krelease) must then apply to this rate-limiting step.A Thermodynamic Analysis That Predicts the Positions of Intrinsic Termination. By treating the reaction described in Eq. 2 as a "virtual" process governed by equilibrium thermodynamics, we may write: DNA + RNA, + polymerase = (ternary complex),. [3] At each template position I, we equate the thermodynamic stability of the ternary complex with its standard free energy of formation (AG'compiex). To calculate AG'compiex we assume the "transcription bubble" model for the structure of the elongation complex (see refs. 4 and 5; Fig. 1 The average value of AG'complex is found to be about -18 kcal/mol at nontermination positions along the DNA template (S). This represents the average free energy that must be added to the ternary complex to reach the state in which AG'.complex 0 kcal/mol. To release the nascent transcript, an activation free energy must also be added to reach the transition state for termination. Thus, at nontermination positions (which probably include >99.9% of all sites on E. coli DNA; see ref. 5), the ternary (elongation) complex should move along the DNA template in a deep potential energy well, elongation should be highly processive, and the probability of spontaneous dissociation of the elongation complex should be very low. These predictions are confirmed by the finding that elongation complexes can be "stalled" (at nontermination positions) by depletion of NTP substrates Abbreviation: TE, termination efficiency. 2307The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

At any template position, the decision to extend the transcript by one residue or to release the nascent RNA represents a kinetic competition between elongation and termination pathways. This competition is discussed in terms of alternative Eyring transition state barriers; changes in termination efficiency correspond to small changes in the relative heights of these barriers. Elongation complexes are stable at nonterminator positions; a model is presented to explain the destabilization of these complexes at intrinsic termination sites. Functionally analogous effects can operate at rho-dependent terminators. Mechanisms for modulation of termination efficiency by regulatory proteins are described.