Structure determination of bacteriophage [phi]21 N-peptide and boxB RNA complex by NMR spectrocsopy
Author(s)Cilley, Christopher D. (Christopher David), 1963-
Massachusetts Institute of Technology. Dept. of Biology.
James R. Williamson.
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The antitermination protein N from [delta] and related E. coli bacteriophage interacts with the nut RNA site and host factors to transform RNA polymerase (RNAP) into a termination resistant transcription complex. The [delta], P22 and [phi]21 phage N proteins share a conserved amino-terminal, arginine-rich region, which confers phage-specific binding to boxB hairpins within the phage nut site RNA. To facilitate nuclear magnetic resonance (NMR) spectroscopy studies of the [phi]21 N-nut RNA complex, we sought a peptide model for the binding of [phi]21 protein to nut RNA. In order to test the specificity of this model, and minimize the size of the complex, we developed a polyacrylamide affinity co-electrophoresis (PACE) assay. This assay is well suited for measuring affinities of small complexes, complexes in rapid equilibrium, and weak affinity protein-RNA interactions. NMR methods were used to determine the solution structure of a 22-amino acid peptide from the amino-terminal domain of [phi]21 in complex with the 24-nucleotide boxB. The boxB RNA hairpin adopts a stem-loop structure with a nine base pair A-form helical stem, and a hexanucleotide loop (5'-CUAACC-3'), with the last four bases continuously stacking on the 3'-half of the hairpin stem. The second nucleotide in the loop (U11) is orientated into the center of the loop, packing against the stacked bases. A core 13-amino acid region of [phi]21 peptide binds as an a-helix and interact predominately with the major groove side of the 5'-half of the ascending upper stem and the loop of boxB. There are a number electrostatic and hydrogen bond interactions with the phosphodiester backbone. There appear to be no significant basespecific interactions. The peptide-RNA interface is defined by surface complementarity of polar and non-polar interactions. Comparison of the [phi]21 complex to the [delta] and P22 structures yields important information about RNA-protein interaction and discrimination. All three peptides bind the RNA stems using polar and non-polar contacts that are almost superimposable. Peptide-loop interactions diverge from this structural similarity. The pentanucleotide loops of [delta] and P22 adopt GNRA-type tetraloop folds with exclusion of one of the five nucleotides. All three peptides show loop-specific interactions. While not a tetraloop, the [phi]21 boxB loop nevertheless adopts a structure very similar to the [delta] loop in shape and exposed surface groups. This structural mimicry may be important for the interaction of the N-boxB complex with host factors in the antitermination complex, particularly NusA.
Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001.In title on t.p., "[phi]" appears as the lower-case Greek letter.Includes bibliographical references (p. 148-159).
DepartmentMassachusetts Institute of Technology. Dept. of Biology.
Massachusetts Institute of Technology