Crosslinking by psoralens and other reagents will be used to map aspects of the secondary and tertiary structure of free and ribosome-bound rRNAs. Initial work will focus on the E. coli 16S rRNA. Of particular interest are contacts between distant regions of the polynucleotide chain. Crosslinks between such regions are analyzed by electron microscopy and gel electrophoresis. Improvements in these techniques should greatly speed the analysis of RNA structure and allow direct comparison of the folded structures of rRNAs in bacteria, organelles and eukaryotes. New rapid techniques will be developed to allow the positions of crosslinks to be localized a the level of the nucleic acid sequence. Crosslinking will also be used to analyze for interactions between the 6 RNAs on a functioning ribosome. We will also develop techniques for examining the presece of knotted topologies in RNAs. Chemical modification, exchange reactions and x-ray scattering will be used to examine the structure of ribosome-bound mRNA. Blotting techniques will be developed to allow crosslinking studies of RNAs without purification. These should have broad applicability to studies on RNA splicing, tumor virus RNA structure and the structure of particular mRNA during transcription, processing and translation. The rationale behind all of these projects is the recent realization that isolated large RNAs can form stable interesting compact folded stgructures. Long distance contacts are uniquely informative n establishing and comparing such structures. Simple screens for such contacts could profoundly influence current research in the biophysical chemistry and molecular biology of regulation, tumor viruses and protein synthesis.
