Molecular recognition of RNA

RNA adopts a variety of molecular structures, from simple single strands to large and complex globular structures containing helices, loops, and bulges arranged together in precise orientations. In order to carry out the varied roles RNAs play in biology, their three dimensional structures must be recognized by proteins, small molecules, and other nucleic acids. Additionally, small RNA elements must recognize each other in order for large RNAs to fold into their proper structures. This thesis will examine two modes of molecular recognition of RNA.; First, I will consider recognition of RNA by proteins. The focus will be on the method employed by the stem-loop binding protein (SLBP) to recognize a stem-loop structure found at the 3′ end of replication dependent histone mRNAs. SLBP, an evolutionarily conserved protein with no known homologues, interacts with the stem-loop in both the nucleus and cytoplasm and mediates nuclear-cytoplasmic transport as well as 3′ end processing of the pre-mRNA by the U7 snRNP. Through this interaction, SLBP mediates the cell cycle dependent regulation of histone levels in higher eukaryotes. I will demonstrate that the SLBP-RNA interaction is highly stable and specific. I will show that the high affinity binding site for SLBP is a 20 nucleotide stem-loop RNA structure found at the 3 ′ end of histone pre-mRNAs, and that SLBP recognizes nucleotides in the stem, loop, and 5′ flanking region of the RNA. I will then elucidate the domain architecture and secondary structure of the SLBP-RNA complex. Together, these results suggest a novel mode of protein-RNA recognition that forms the core of a ribonucleoprotein complex central to the regulation of histone gene expression.; Next, I will examine the recognition of RNA by RNA. The predominant RNA-RNA tertiary contact is the A-minor motif. In this interaction, an adenosine residue from one RNA structural element recognizes the minor groove of a neighboring RNA helix. By a combination of biochemical and structural techniques, my work will demonstrate that the adenosine involved in an A-minor interaction is specific for Watson-Crick minor groove geometry. This work will provide a thermodynamic and structural basis for the specificity of the motif responsible for mediating the majority of the interhelical tertiary interactions in complex RNAs.