Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors

Abstract

Efficient and specific interactions between proteins bound to the same DNA molecule can be dependent on the length of the DNA tether that connects them. Measurement of the strength of this DNA tethering effect has been largely confined to short separations between sites, and it is not clear how it contributes to longrange DNA looping interactions, such as occur over separations of tens to hundreds of kilobase pairs in vivo. Here, gene regulation experiments using the LacI and λ CI repressors, combined with mathematical modeling, were used to quantitate DNA tethering inside Escherichia coli cells over the 250-to 10,000-bp range. Although LacI and CI loop DNA in distinct ways, measurements of the tethering effect were very similar for both proteins. Tethering strength decreased with increasing separation, but even at 5-to 10-kb distances, was able to increase contact probability 10-to 20-fold and drive efficient looping. Tethering in vitro with the Lac repressor was measured for the same 600-to 3,200-bp DNAs using tethered particle motion, a single molecule technique, and was 5-to 45-fold weaker than in vivo over this range. Thus, the enhancement of looping seen previously in vivo at separations below 500 bp extends to large separations, underlining the need to understand how in vivo factors aid DNA looping. Our analysis also suggests how efficient and specific looping could be achieved over very long DNA separations, such as what occurs between enhancers and promoters in eukaryotic cells. j factor | TPM | promoter-enhancer I nteractions between proteins bound to separate sites on the same DNA molecule are critical in gene regulation and other DNA processes (1-4). The DNA separation between functionally interacting sites ranges from a few base pairs to hundreds of kilobase pairs, as in the case of some eukaryotic enhancers and their promoters. At short separations, it is clear that the DNA acts as a tether that keeps one site in the vicinity of the other so that the proteins at one site can find the other site in 3D space more efficiently than if they were free in solution (Fig. 1). Tethering is also a way to provide specificity because it aids interaction with linked sites but not unlinked sites. However, as the separation between the sites increases, this tethering effect becomes weaker, and it is not understood how the DNA linkage between widely separated sites, for example, between enhancers and promoters, provides the efficiency and specificity required for proper regulation.The effect of DNA tethering can be quantified by the factor j LOOP (M), the effective molar concentration of one site on the DNA relative to the other, or as the free energy of the DNA looping reaction ΔG LOOP (Fig. 1) (5-9). These parameters are interconvertible: ΔG LOOP = -RTlnj LOOP (kcal/mol; the reference j is 1 M). The formation of a naked DNA loop is in itself an energetically unfavorable reaction (ΔG LOOP is positive) under physiological conditions due to the enthalpic cost of DNA bending and twisting (particularly important for s...

Quantitation of interactions between two DNA loops demonstrates loop domain insulation in E. coli cells

“…3 B and C), with loop sizes ranging from 1,200 to 1,800 bp. The smallest distance between operators was 300 bp, which is well beyond the apparent 20-to 45-bp persistence length of DNA in E. coli (24,50).…”

“…Previously we used in vivo reporter assays and statisticalmechanical modeling to extract key DNA-looping parameters for LacI and CI DNA loops (24), allowing calculation of the fraction of the time (averaged over many cells) that the DNA is in a looped state, which we term F. The rationale of this approach is shown in Fig. 2.…”

“…For CI, the fraction of DNA looping can be obtained from measurements of CI regulation of the phage PRM promoter (24,27,48). At low CI concentrations, CI tetramers assemble at the OL and OR sites, and these DNA-bound tetramers can form an OL-OR DNA loop by CI octamerization (Fig.…”

“…1A), because the efficiency of contact increases as the length of the DNA tether between the sites shortens (20)(21)(22)(23)(24). Promoter-tethering elements in Drosophila that allow activation by specific enhancers over long distances are proposed to form DNA loops between sequences near the enhancer and the promoter (18,25).…”

Proteins mediating DNA loops effectively block transcription

“…of incubation (see Materials and Methods), LacI has established a dynamic equilibrium with operator sites along DNA molecules. However, 7.5 nM LacI fairly saturates Os operators ( K d  < 1 × 10 −14 M in similar K + conditions29), and the estimated tether‐augmented, effective concentration ( J value) of LacI is expected to be in the range of 4 to 100 nM for a 400 bp loop (data not shown30). Therefore, operator occupancy is expected to be very high throughout the 60 s incubation.…”