Interaction of the Bacillus stearothermophilus ribsosomal protein S15 with rRNA

The ribosomal RNA binding site of Bacillus stearothermophilus ribosomal protein S15 (BS15) was analyzed using synthetic RNA oligonucleotides derived from the 16S rRNA central domain. Native gel electrophoresis mobility shift assays demonstrate that BS15 can specifically interact with an RNA oligonucleotide containing nucleotides 585-756 (helices 20-23) of 16S rRNA with an apparent dissociation constant of 35 nM. A series of deletion mutants of the rRNA fragment that contains the BS15 specific binding site was tested for their capacity to bind protein using a competition binding assay, yielding a 61 nucleotide RNA that is capable of binding BS15 with wild type affinity. The major determinant of the BS 15-rRNA interaction is a three-way junction between helices 20, 21, and 22, while helix 23 (nucleotides 673-733 of 16S rRNA) was dispensable for high affinity binding. Kinetic measurements of the dissociation and association rate of the bimolecular complex between BS 15 and various minimal rRNA binding sites demonstrate that the kinetic and thermodynamic properties of this interaction were not altered as a result of the deletions. The sequence and structural determinants for the RNA in the BS15-rRNA interaction have been probed using site-directed mutagenesis, chemical modification interference, and iodine footprinting of phosphorothioate RNA. Mutations and RNA modifications that interfere with protein binding cluster in two distinct regions, one containing an internal loop and the other containing a three-way junction. Projection of this data onto a three-dimensional model illustrates that BS 15 is likely to interact with the minor groove along an extended face of helix 22. This minimized BS15-rRNA interaction has been examined using NMR spectroscopy. As a prelude to structural studies of this large protein-RNA complex by NMR, a general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs has been developed. For 15N-labeling, E. coli is grown on 15N-ammonium sulfate, whereas for 2Hand 13C-labeling, Methylophilus methylotrophus is grown on 13C-methanol, which is more economical than 13C-glucose. While the 30.5 kDa BS15-Fr15 complex yields high quality homonuclear spectra, indicative that this 13 complex is well-behaved under NMR conditions, the introduction of C into the RNA causes severe line broadening in heteronuclear 3D-NMR spectra, limiting the applicability of these types of experiments. Thesis Supervisor: James R. Williamson Associate Professor of Chemistry Title: This thesis is not the accomplishment of a single person, but rather a synthesis of the ideas, experience, and passion of many people. While I reserve these few pages to express my appreciation for the support of many people, my ultimate gratitude is reflected in the pages that follow. First, and foremost, I am indebted to Jamie. The S 15 project was founded after he challenged me to go off and find an interesting RNA-protein interaction to study. His support for this project, even during the fabled dark year, has always been enthusiastic and wholehearted. During the course of this project, he has allowed me the intellectual and scientific freedom to explore this project, which I believe, more than anything else, has contributed to a rewarding graduate career. However, without his strong guidance and unyielding belief that this project must work, the S15 story would have been left on the cutting room floor years ago. In the early years of my graduate career, the Williamson lab was graced by the presence of Jody Puglisi. There are not enough words to accurately describe the impact Jody had on molding the ideas and nature of this lab. Jody brought to this laboratory a working and practical knowledge of RNA biochemistry and NMR that profoundly impacted all of our studies (as evidenced by the fact that the most used document in this lab for the last five years has been his thesis). I hold the pearls of wisdom about RNA, science, and life in general he has given me over the years in the highest regard. The people within the Williamson laboratory during my career have contributed much to this thesis. During my time here, I have had the pleasure to interact with a group of people that brought together an impressive array of skills. Pat Zarrinkar has provided many fruitful discussions about RNA techniques and biochemistry. Furthermore, without him, many of the day-to-day aspects of the lab would have fallen into complete chaos. While his vast organizational skills have provided us with many hours of amusement at his expense, there has not been a single person whose life in this lab has not been made a lot easier by his efforts. Similarly, Dan Treiber has made many positive contributions to this work with his strong working knowledge of molecular biology and protein-nucleic acid interactions. All of the NMR work in this thesis has been with the help and training of L.I----·-~----_ls~---r _h--~-____~r i~mn-_~=-r----~ -