PROTEIN &NUCLEIC ACID FOOTPRINTING OF RNA POLYMERASE

As preliminary studies prior to time-resolved nucleic acid and protein footprinting, we are biochemically establish the contacts between thumb and the template-primer. This will be done by comparing the hydroxyl radical footprinting of HIV-1 RT or HIV-1 RT-nevirapine complex on synthetic DNA template-primer duplexes. The thumb moves away from the template-cleft when bound to nevaripine. Thus, sites protected by apo-RT should be de-protected in the presence of nevaripine during hydroxyl-radical footprinting. Once we have established the portions of the template-primer that are protected specifically by the thumb sub-domain, we will perform x-ray footprinting during controlled polymerization. Omitting one or more dNTPs in the reaction will allow controlled polymerization. Since RT is known to catalyze the polymerization at a rate of many milliseconds per monomer, it will be possible to measure the time-dependent changes Hin thumb position via detection of protection of template-primer Hand/or the thumb or lack of it.