Protein-DNA Interactions

The characterization of the protein-DNA interactions usually requires three levels of analysis:

GENETIC

• determination of the nucleotide sequence of the protein-binding region and the identification of sequence changes that confer a mutated phenotype.

 

BIOCHEMICAL

• identification of potential protein-DNA contacts using a variety of footprinting and protection experiments such as DNaseI or hydroxyl radical footprinting, and methylation protection or ethylation protection experiments.
  
• quantification of binding using gel-shift techniques
  
  

PHYSICAL

• Analysis of specific interactions in protein-target sequence fragment co-crystals.

General Approach

The underlying philosophy behind footprinting or protection experiments is the same even though the information that they yield is different. There are 2 general principles:

• The DNA fragment is labelled at ONE end only
• Each DNA fragment is hit once only

 

DNA labelling techniques will label both ends of a DNA fragment equally. However, use of a restriction enzyme that cuts close to one end will generate two fragments of unequal size that can be separated and purified.

Reaction conditions must be carefully controlled so that the chosen reagent (DNaseI, DMS) attacks only once in ~300 bp. This means in practice that reactions are brief (limit the time) and that reagent concentrations are low.

DNase I Footprinting

This technique allows one to determine the overall span of DNA that is bound by a protein of interest. It doesn't give one any detailed information on base pair recognition. However, by examining the footprints of multiple proteins, one can get an idea whether their binding sites overlap, whether they are mutually exclusive, or whether they seem to assist one another in binding.

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A good example of the use of DNaseI footprinting is found in a paper by Xiao-Yong Li and William R. McClure titled "Characterization of the Closed Complex Intermediate Formed during Transcription Initiation by Escherichia coli RNA Polymerase" and published in J Biol Chem, 273(36): 23549-23557, 1998.

 

These authors studied binding of RNA polymerase to the bacteriophage l prmup-1 D265 promoter under conditions in which only closed complexes (and very few open complexes) would form. Quoting from the paper:

"Since only partial closed complex formation could be achieved under the experimental conditions, we have determined the fractional protection at each position by carrying out quantitative DNase I footprinting at several RNA polymerase concentrations. The analysis of these data permits the protection pattern of fully-formed closed complexes to be determined. The results show that the closed complex is distinct from that of the open complex, indicating significant changes in the RNA polymerase-promoter interaction during open complex formation."

 

Two Protocols

Protection -vs- premodification

Methylation Protection

DNaseI footprinting doesn't give one any detailed information on base pair recognition. DNaseI is a fairly large protein that needs to access DNA phosphodiester bonds where. However, use of several smaller chemical probes allows for a more specific picture to be obtained. The most common reagenst are dimethylsulfate (which probes contact with purines, particularly G), ethynitrosourea (which probes contact with the phosphodiester backbone), and hydroxyl radicals (which probe...).

[MvH4A-1]

Dimethylsulphate reacts readily with purines to form N7-methylguanine or N3-methyladenine. This reaction is the basis of the original Maxam-Gilbert sequencing reaction for purines. The reaction protocol is carried out much as for DNase footprinting. The results also appear similar except that, for the most part, the effect of protein binding on only G residues is discovered.

The N7 position of guanine is located in the major groove of a double helix so protection experiments will generally reveal the proximity of protein side-chains to specific bases in the major groove. The N3 position of adenine, by contrast, is located in the minor groove of a double helix. Effects on A residues are therefore indicative of proteins binding to DNA via the minor groove.

The figure at right (from Xiao-Yong Li and William R. McClure (1998) Stimulation of Open Complex Formation by Nicks and Apurinic Sites Suggests a Role for Nucleation of DNAMelting in Escherichia coli Promoter Function. J Biol Chem, 273(36): 23558-23566.) shows the effects of RNA polymerase on methylation of the bacteriophage l prmup-1 D265 promoter open complex. Note that methylation of some positions is enhanced by RNA polymerase binding while methylation is inhibted at other positions.

 

Ethylation Interference

While, in principle, any of the chemical exploration experiments can be carried out using either a protection or a premodification protocol, in practice they are not. Methylation experiments are usually carried out to probe protection of a base by the presence of a protein. Ethylation modification, by contrast, are usually carried out before protein binding is tested. In this protocol, the presence of the modification may alter the binding affinity of the protein.

Ethylation interference can identify critical DNA phosphate backbone contacts for protein interactions. Such contacts are important because the electrostatic interactions provide the binding energy of the interaction. Furthermore, since the phosphate backbone is relatively uniform regardless of the sequence of bases, ethylation interference is one of the few techniques available to investigate non sequence specific interactions.

The figure at right (from: Richard J. Noel Jr. and William S. Reznikoff (2000) Structural Studies of lacUV5-RNA Polymerase Interactions in Vitro; ETHYLATION INTERFERENCE AND MISSING NUCLEOSIDE ANALYSIS. J Biol Chem, 275(11): 7708-7712.) show the effect of ethylation on binding of RNA polymerase to the lacUV5 promoter. You will need ot look closely to see some of these effects; or, you can view a densitometric analysis in the online manuscript.

 

 

 

Hydroxyl Radical Interference

Hydroxyl radical experiments (aka: missing nucleoside experiments) can identify specific contacts to bases in DNA. Hydroxyl radical treatment results in oxidative degradation of deoxyribose and leads to destruction of the phosphate backbone and loss of the nucleoside at the position of modification. The products are a 5'-phosphoryl group at one end and an equal mix of 3'-phosphoryl and 3'-phosphoglycolic acid at the other end. The modification results in base loss, breakage of the phosphate backbone, and the addition of two negative charges to the backbone.

This technique offers an advantage over other interference and protection techniques because it removes structure rather than altering existing structure. Furthermore, the missing nucleoside approach is more general than methylation interference because hydroxyl radicals modify all positions relatively equally.

Overall

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