NUCLEIC ACID STRUCTURE AND PROTEIN INTERACTIONS

Yale University, New Haven CT

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Abstract

A combination of molecular biological and biophysical methods will be used to elucidate specific aspects of RNA and DNA function in structural and mechanistic terms. Problems concerned with RNA structure and interactions include characterization of the rapid switch in conformation which we have discovered in the 5'-half of Trypanosoma spliced leader RNAs. Relaxation kinetic, NMR, and enzymatic/chemical footprinting methods will be used to characterize the wild type RNA and mutants which lock the structure in one of the forms which are in rapid (~100 musec) equilibrium in the wild type RNA. NMR studies will focus initially on the imino proton spectra, since according to our working model the structural switch involves two competing and quite different secondary structures. We propose also to use NMR, kinetic and molecular biological methods to characterize the nature of the complex between the loops of two small hairpin helices which serve as models for the sense-antisense RNA interaction that controls replication in ColE1 plasmid. Similar methods will be used to investigate the structure and origins of specificity in the interaction of the RNA loop-loop complex with the control protein ROM. Projects which focus on DNA will include completion of electrophoretic experiments on the contribution of non-A- tract sequences to DNA bending. Multimeric oligonucleotides having variable sequence repeats will be studied in order to disentangle the influence of changes in curvature from the effect of changing the helical repeat. DNA topology in E. coli CAP protein and CAP-polymerase complexes will be investigated by measuring the rate of cyclization of specially designed DNA constructs in the presence and absence of bound proteins. A first objective is to determine the bend angle induced in DNA by CAP binding. Cyclized DNAs will also be used to determine the sequence- dependent free energy of bending by measuring the relative binding affinity of DNA molecules which are pre-bent by cyclization. Finally, we will amplify and determine the strongest nucleosome binding sequences from bulk chromatin, with the objective of clarifying the sequence signals which encode strong nucleosome positioning.