PROTEIN &NUCLEIC ACID FOOTPRINTING OF RNA POLYMERASE

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date10/1/959/30/02

Funding

  • National Center for Research Resources

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  • ALBERT EINSTEIN CENTER FOR SYNCHROTRON BIOSCIENCES

    Blasie, K. J., Chance, B., Chance, M. R., Almo, S., Anderson, R., Anni, H., Brenowitz, M. D., Burke, J., Chen, T., Crothers, D., Dauter, Z., Davies, D., Downard, K. M., Fisher, T., Franzen, S., Goldsmith, S., Heiney, P. A., Holmes, K. C., Hurley, J., Hurley, J., Ji, X., Kovacs, K., Kravtsov, I., Lee, C. C., Maleknia, S. D., Marinkovic, N., Maroney, M. J., Maroney, M., Miller, L. M., Morris, A., Pedersen, L., Penner-Hahn, J. E., Powers, L. S., Pyle, A. M., Que, L., Que Lawrence, J., Sagi, I., Scarrow, R. C., Scheuring, E., Seeman, N. C., Silva, A., Snyder, G., Sonenberg, N., Sperber, E., Studier, B., Sullivan, M. D., Sun, P., Toomey, J. R., Tran, A., Vanamee, E. S., Wlodawer, A., Yan, H., Bubacco, L., Caffrey, M., Chance, M. R., Kallenbach, N., Sharpe, L., Sinclair, R., Woodson, S., Xie, A. & Yue, K.

    8/1/858/31/03

    Project: Research project