Arnold Revzin

The use of gel electrophoresis to detect and study nucleic acid— protein interactions

Abstract Gel electrophoresis is now a tool in the repertoire of every laboratory that works with proteins or nucleic acids. In this article we describe a simple and convenient extension of this technique for the study of nucleic acid-protein systems, especially the interactions involved in the regulation of gene expression.

The gene encoding cytochrome c oxidase subunit II from Rhodobacter sphaeroides; comparison of the deduced amino acid sequence with sequences of corresponding peptides from other species

The gene (coxII) encoding subunit II of Rhodobacter sphaeroides cytochrome c oxidase (cytochrome aa3) has been isolated by screening a genomic DNA library in phage lambda with a probe derived from coxII of Paracoccus denitrificans. A 2-kb fragment containing coxII DNA was subcloned into the phage M13mp18 and the sequence determined. The 2-kb insert contains the entire coding region for coxII gene, including the ATG start codon and a TGA stop codon. The deduced amino acid (aa) sequence of subunit II of R. sphaeroides shows regions of substantial homology to the corresponding subunit of the bovine mitochondrial oxidase (63% overall) and P. denitrificans oxidase (68% overall). The postulated redox-active copper ion (CuA) binding site involving two Cys and two His residues (as well as an alternative Met residue) is conserved among these species, along with four invariant acidic aa residues (two Asp and two Glu) that may be involved in interactions with cytochrome c, and a region of aromatic residues (Tyr-Gln-Trp-Tyr-Trp-Gly-Tyr-Glu-Tyr) which is postulated to play a role in electron transfer. Hydropathy profile analysis suggests that while the bovine COXII secondary structure contains two transmembrane helices, the R. sphaeroides subunit II has a third such helix that may function as part of a signal sequence, as suggested for P. denitrificans.

Interference and Missing Contact Footprinting

Publisher Summary This chapter discusses interference and missing contact footprinting. The interference and missing contact techniques for probing DNA-protein interactions differ from other footprinting methods as they involve modification of the DNA before addition of the protein of interest, rather than measuring protection of a DNA region by bound protein from a reagent that would otherwise attack that sequence. The interference and missing contact approaches may not always lend themselves to analysis of multiprotein complexes, because a modification that obstructs binding of one of the proteins may prevent the binding of the other participants as well. The combination of interference and missing contact techniques provides a wealth of detail about DNA-protein interactions that may not be accessible by other footprinting-type methods. The interference and missing contact methods have helped to elucidate the properties of numerous purified DNA-protein systems. Nevertheless, the interference and missing contact techniques provide a wealth of valuable data, which often will form the basis for future studies on DNA-protein complexes of interest.

Exonuclease III Digestion

Publisher Summary This chapter provides an overview on exonuclease III Digestion. The processive enzyme exonuclease III from Escherichia coli can be used to locate the binding site of a protein on a native DNA fragment. Exonuclease III can be obtained from a variety of suppliers, including United States Biochemicals, Boehringer Mannheim. A difficulty with the exonuclease III technique is that the efficacy of the enzyme is not always uniform, in that certain phosphodiester bonds in the DNA sequence may be resistant to cleavage. Exonuclease III may pause at points such as it moves along the DNA, and if it dissociates from the fragment there extra-neous bands will be generated. Exonuclease III can be obtained from a variety of suppliers, including United States Biochemicals, Boehringer Mannheim.