In Chapter 2 we discussed how a histone bound to DNA can protect the nucleotides it contacts from DNAse cleavage of phosphodiester bonds. Biochemical methods similar to those DNAse footprinting experiments can even identify interactions between specific amino acid residues and specific bases. In some cases the biochemical approaches provide as much resolution as X-ray crystallography.

First, consider how to test a guess of a specific residue-base interaction. In this approach a correct guess can be confirmed, but a poor guess yields little information. The idea is that if the amino acid residue in question is substituted by a smaller residue, say a glycine or alanine, then the former interaction between the residue and the base will be eliminated. As a result, the strength of the protein’s binding becomes independent of the identity of the base formerly contacted, whereas the wild-type unsubstituted protein would vary in its affinity for the DNA as the contacted base is varied (Fig. 1).

Fig1. How replacing a DNA contacting residue of a protein with a smaller residue like alanine or glycine makes the protein indifferent to the identity of the base that formerly was contacted by the residue.

The work required for this missing contact experiment is high. Genetic engineering techniques must be used to alter the gene encoding the protein. Similarly, both the wild-type sequence and a sequence for each base being tested for contacting must also be synthesized. Finally, the protein must be synthesized in vivo, purified, and its affinity for the various DNAs tested.

The testing for specific contacts can be streamlined. Instead of having to guess correctly both the base and the residue, the technique finds whatever base is contacted by a residue. As before, a variant protein is made containing a glycine or alanine substitution in the residue suspected of contacting the DNA. The DNA sample is probed biochemically to find all the residues contacted by the wild-type protein and by the mutant protein. The difference between the set of bases contacted by the wild-type protein and the mutant protein is the base or two that are normally contacted by the altered residue. The procedure for locating the bases which contact the protein is more fully described in Chapter 10.

مواضيع ذات صلة

Fidelity of Aminoacylation

Activation of Amino Acids During Protein Synthesis

GENE TRANSCRIPTION

كيف نكشف عن هوية الشخص الجينية

طرق توسيم المجسات (labeling of probes) للكشف عن الجين الهدف

الـ PCR المتزامن الكمي (quantitative Real time PCR)

الـ PCR ذو البداية الحارة (hot start PCR)

Salt Effects on Protein-DNA Interactions

Structures of DNA-Binding Proteins

Secondary Structure Predictions

Calculation of Protein Tertiary Structure

RNA Heterogeneity