For nucleic acid oligomers with variable chain lengths, the salt concentration ([salt]) dependences of the denaturation temperature (T(m)) and of the free energy of helix formation at 37 degrees C (Delta) are predicted using nonlinear Poisson-Boltzmann (NLPB) calculations. Analysis of experimental data reveals that the ratio of the [salt] derivative of melting temperature (ST(m) = dT(m)/d log[salt]) to the value for a polymer with the same base composition (ST(m)/ST(m, infinity)) is independent of base composition but strongly dependent on the number of DNA charges (/Z/) below approximately 8 bp for two-strand helices (formed from association of two complementary strands) and below approximately 18 bp for hairpin helices (formed from folding of one self-complementary strand). We interpret these ST(m)/ST(m, infinity) ratios in terms of the ratio of thermodynamic ion release from the oligomer (Deltan(u), per charge) to that from the same oligomer embedded in polymeric DNA (Deltan(u, infinity), per charge). Experimental values of ST(m)/ST(m, infinity) and its dependence on /Z/ are in good agreement with NLPB predictions for a preaveraged (essential structural) model of DNA. In particular, the NLPB calculations describe the stronger /Z/ dependence of ST(m) observed for melting of oligomeric hairpin helices than for melting of two-strand helices. These calculations predict an experimentally detectable (>or=10%) difference between ST(m) and ST(m, infinity) which increases strongly with decreasing length for two-strand helix lengths of <15 bp and for hairpin helix lengths of <30 bp. From NLPB values of Deltan(u)/Deltan(u, infinity), we predict Delta as a function of [salt] and /Z/. Predictions of thermodynamic and thermal stabilities of oligomeric helices as functions of length and [salt] are consistent with and represent a significant refinement of the average oligomer salt effect currently in use in nearest neighbor stability predictions.

FRET (fluorescence resonance energy transfer) between far-upstream (−100) and downstream (+14) cyanine dyes (Cy3, Cy5) showed extensive bending and wrapping of λPR promoter DNA on Escherichia coli RNA polymerase (RNAP) in closed and open complexes (CC and OC, respectively). Here we determine the kinetics and mechanism of DNA bending and wrapping by FRET and of formation of RNAP contacts with −100 and +14 DNA by single-dye protein-induced fluorescence enhancement (PIFE). FRET and PIFE kinetics exhibit two phases: rapidly reversible steps forming a CC ensemble ({CC}) of four intermediates [initial (RPC), early (I1E), mid (I1M), and late (I1L)], followed by conversion of {CC} to OC via I1L. FRET and PIFE are first observed for I1E, not RPc. FRET and PIFE together reveal large-scale bending and wrapping of upstream and downstream DNA as RPC advances to I1E, decreasing the Cy3−Cy5 distance to ∼75 Å and making RNAP–DNA contacts at −100 and +14. We propose that far-upstream DNA wraps on the upper β′-clamp while downstream DNA contacts the top of the β-pincer in I1E. Converting I1E to I1M (∼1 s time scale) reduces FRET efficiency with little change in −100 or +14 PIFE, interpreted as clamp opening that moves far-upstream DNA (on β′) away from downstream DNA (on β) to increase the Cy3−Cy5 distance by ∼14 Å. FRET increases greatly in converting I1M to I1L, indicating bending of downstream duplex DNA into the clamp and clamp closing to reduce the Cy3−Cy5 distance by ∼21 Å. In the subsequent rate-determining DNA-opening step, in which the clamp may also open, I1L is converted to the initial unstable OC (I2). Implications for facilitation of CC-to-OC isomerization by upstream DNA and upstream binding, DNA-bending transcription activators are discussed.