Real-Time Single-Molecule Studies of RNA Polymerase–Promoter Open Complex Formation Reveal Substantial Heterogeneity Along the Promoter-Opening Pathway

Abstract

Graphical abstract

Transient DNA binding to gapped DNA substrates links DNA sequence to the single-molecule kinetics of protein-DNA interactions

“…The biotinylated DNA constructs were immobilised at ~0.2 μm -2 density via neutravidin, inside the wells of silicon gaskets, on plasma-cleaned coverslips coated with polyethylene-glycol (PEG; (4, 17)). We then added 20 μl of 50 pM gapped-DNA constructs in Milli Q, incubated for 10 s, and washed three times with 200 μl PBS.…”

“…The fluorescent probe labelled protein (Alexa647-CAP) was purified using a G25 Sephadex gel filtration column pre-equilibrated with buffer B (40 mM Tris-HCl pH 8, 100 mM NaCl, 0.1 mM TCEP and 5% glycerol), followed by buffer exchange (5 cycles of concentration to 0.5 ml volume and dilution with 5 ml buffer B) using a 3 kDa MWCO Amicon Ultra-15 centrifugal ultrafilter (Millipore), and the purified Alexa647-CAP was stored in aliquots at -80 o C. Sample preparation for real-time transient DNA binding. The biotinylated DNA constructs were immobilised at ~0.2 µm -2 density via neutravidin, inside the wells of silicon gaskets, on plasma-cleaned coverslips coated with polyethylene-glycol (PEG; (4,17)). We then added 20 µl of 50 pM gapped-DNA constructs in Milli Q, incubated for 10 s, and washed three times with 200 µl PBS.…”

“…On the other hand, single-molecule analysis of sequence-specific protein-DNA and protein-RNA interactions can uniquely address issues of sample heterogeneity, while offering the ability to monitor interactions and associated conformational changes in real-time (4–6, 10, 11). Such methods include single-molecule fluorescence, force-based methods, and nanopore-based methods (10).…”

“…However, the use of genome sequencing approaches to dissect protein interactions with nucleic acids is complicated by ensemble averaging. Such approaches cannot report directly on the kinetics of protein-DNA interactions, especially when these interactions involve several steps that go beyond relatively simple binding-unbinding equilibria, as in the case of multi-step reactions in gene repair, editing, replication, and transcription, all of which feature transient on-pathway or off-pathway intermediates (4)(5)(6)(7)(8)(9). NGS methods also typically rely on DNA amplification, introducing the possibility of base errors.…”

Evidence for a compact σ70conformationin vitroandin vivo

“…Then, in the context of the RNAP holoenzyme, proper binding of the σ 70 subunit to a promoter initially forms the RNAP-promoter closed (RPC) complex, where σR4.2 binds the -35 promoter element, σR2.3 and σR2.4 bind the -10 promoter element, σR1.2 binds the promoter discriminator sequence, σR3 interacts with the -10 upstream extended element and the C-terminal domain of the α subunit dimer of RNAP also interact with the promoter UP element (5). The formation of the RPC is then followed by a cascade of DNA isomerization events (6)(7)(8)(9)(10), which end with a stretch of 10-12 bases of promoter DNA right upstream to the transcription start site that melts and forms the DNA transcription bubble, stabilized within the RNAP-promoter open (RPO) complex (11,12). Importantly, throughout transcription initiation complex, σ 70 exhibits an extended structure, whereby σR2 and σR4 are far apart from each other, to facilitate proper promoter binding.…”