“ Sequence-specific DNA-binding proteins play a key role in many fundamental bio
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Question
“ Sequence-specific DNA-binding proteins play a key role in many fundamental biological processes, such as transcription, DNA replication and recombination. Very often, these DNA-binding proteins introduce structural changes to the target DNAbinding sites including DNA bending, twisting or untwisting and wrapping, which in many cases induce a linking number change (Lk ) to the DNAbinding site. Due to the lack of a feasible approach, Lk induced by sequence-specific DNA-binding proteins has not been fully explored. In this paper we successfully constructed a series of DNA plasmids that carry many tandem copies of a DNA binding site for one sequence-specific DNA-binding protein, such as O, LacI, GalR, CRP and AraC. In this case, the protein-induced Lk was greatly amplified and can be measured experimentally. Indeed, not only were we able to simultaneously determine the protein-induced Lk and the DNA-binding constant for O and GalR, but also we demonstrated that the protein-induced Lk is an intrinsic property for these sequence-specific DNA-binding proteins. Our results also showed that protein-mediated DNA looping by AraC and LacI can induce a Lk to the plasmid DNA templates. Furthermore, we demonstrated that the protein-induced Lk does not correlate with the protein-induced DNA bending by the DNA-binding proteins.” NAR 38:3643.
1. What is the definition of Lk?
2. If DNA is negatively supercoiled, what is the value of Lk?
3. In general, how do DNA-binding proteins, such as O, LacI, GalR, CRP and AraC, effect Lk and DNA supercoiling?
4. How might DNA supercoiling, and the DNA binding proteins (O, LacI, GalR, CRP and AraC), effect transcription?
Explanation / Answer
1]The linking number of a DNA molecule rigorously specifies the number of helical turns in a closed-circular DNA, in the absence of any supercoiling. Linking number is a topological property because it does not vary when double-stranded DNA is twisted or deformed in any way, as long as both DNA strands remain intact.
2]The linking number for a closed-circular DNA is always an integer. By convention, if the links between two DNA strands are arranged so that the strands are interwound in a right-handed helix, the linking number is defined as positive (+). Conversely, for strands interwound as a left-handed helix the linking number is negative (-). Given that left-handed Z-DNA occurs only rarely, negative linking numbers are not encountered in studies of DNA.Negatively supercoiled DNA will have a Lk as 0.
3,4]Sequence-specific DNA-binding proteins play essential roles in many fundamental biological events such as DNA replication, recombination, and transcription. One common feature of sequence-specific DNA-binding proteins is to introduce structural changes to their DNA recognition sites including DNA-bending and DNA linking number change (Lk).DNA-binding proteins also result in DNA unwinding or linking number changes (Lk) to their recognition sites.
E. coli LacI is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. This tetrameric protein specifically binds to the O1, O2 and O3 operators of the lac operon and forms a DNA loop to repress transcription from the lac promoter.LacI forms a topological barrier upon binding to the O1 and O2 operators of the lac promoter at their native positions and constrains three negative supercoils within the 401-bp DNA loop
CRP–cAMP complex might significantly unwind the DNA binding site according to a crystal structure of the CRP–cAMP complex.CRP was able to induce a substantial Lk to its binding site using tandem copies of a CRP recognition sequence if the CRP binding sites match the helical phase of the DNA bends
O is the replication initiator of bacterial phage and specifically binds to the four repeating sequences (iterons) of replication origin (Ori). Upon O binding, Ori wraps around O protein and forms a nucleoprotein complex called “O-some”. As a result, the AT-rich DUE is unwound and ready for subsequent replication initiation. Wrapping DNA around the O protein should cause DNA unwinding or a Lk in the replication origin.
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