We have constructed (and continue to develop) a laser-based facility for time-resolved fluorescence spectroscopy of biomolecules. This facility provides rapid collection and analysis of luminescence data related to macromolecular size, flexibility, folding and structural fluctuations. Our time-correlated laser fluorometer was used to study the folding and dynamics of several proteins. We focused most of our effort on the DNA- binding proteins that control the transcription of DNA blueprints into the """"""""field copies"""""""" (m-rna) used to build cells. We used fluorescence to measure distances between proteins and the sections of DNA they control, to look at the wobbling of proteins in the complex, and to reveal internal changes in the protein that accompany binding. We studied """"""""oct-pou"""""""", a bipartite factor able to bind two different classes of DNA control sites and speed transcription 100-fold. We also studied TFIIIA, a eukaryotic factor that controls the synthesis of 5S-RNA (a component in the protein """"""""forge"""""""" that carries out blueprint instructions). We found it to be more compact in solution than when its' nine """"""""zinc fingers"""""""" grasp DNA. Our studies of these and several similar factors are aimed at learning the general process of transcription control. This year we laid the spectroscopic foundation down for simultaneous study of several related transcription factors. We began to study other DNA-protein problems, also. We started work on HIV integrase and beta-polymerase. The former incorporates AIDS into human DNA, and the latter repairs UV-damaged DNA. This year we also continued efforts to adapt our laser instruments to the imaging of tissues. Tissue diffuses even red light rapidly, but our subnanosecond timing lets us examine the """"""""shock wave"""""""" of brightness engulfing tissues. This may someday lead to practical, noninvasive tissue imaging devices that compete with MRI. In particular, we have developed an electronic laser scanning system that can locate - and more importantly, reveal the color of - BB-sized objects almost an inch inside tissue. Thus, we hope to develop a useful adjunct to mammography.
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