IDENTIFICATION OF A TUS PROTEIN SEGMENT THAT PHOTO-CROSS-LINKS WITH TERB DNA AND ELUCIDATION OF THE ROLE OF CERTAIN THYMINE METHYL-GROUPS IN THE TUS-TERB COMPLEX USING HALOGENATED URACIL ANALOGS

Six potential hydrophobic sites of the Tus-TerB complex were analyzed using bromodeoxyuridine and iododeoxyuridine as isosteric analogues of thymine. Analogues were incorporated at individual sites, and dissoci ation rates were measured in 150 mM potassium glutamate, pH 8.0, using a nitrocellulose filter assay. These halogenated analogues serve as a probe of the environment in which they reside. Our measurement reveal ed at least two types of responses. Three sites showed increases in st ability with the introduction of the bromo and iodo derivatives. The e nhanced stability is proposed to result from polar or charged molecule s in the vicinity of the halogenated analogues through dipole-dipole, dipole-ion, or dipole-induced dipole interactions. The other three sit es exhibited the opposite trend, being destabilized by the introductio n of these analogues. The destabilizing effects are attributed to a hy drophobic environment which cannot accommodate polar molecules. The ph otoreactivity of these analogues was utilized to specifically cross-li nk the Tus protein and TerB DNA. Using the substitution of bromodeoxyu ridine at position 8 in the TerB DNA, Tus protein was covalently attac hed to the DNA, and by trypsin digestion a fragment of Tus was isolate d. Sequencing of the peptide fragment revealed a segment that matched the amino acid composition from 122-139 of the Tus protein.