2026-09-23T02:37:16Zhttps://dspace.mit.edu/server/oai/requestoai:dspace.mit.edu:1721.1/85872022-01-13T07:54:15Zcom_1721.1_7582com_1721.1_7581col_1721.1_131022
James R. Williamson.
Cilley, Christopher D. (Christopher David), 1963-
Massachusetts Institute of Technology. Dept. of Biology.
Massachusetts Institute of Technology. Department of Biology
2005-08-23T21:30:24Z
2005-08-23T21:30:24Z
2001
2001
http://hdl.handle.net/1721.1/8587
49265221
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).
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.
by Christopher D. Cilley.
Ph.D.
159 p.
11899882 bytes
11899639 bytes
application/pdf
application/pdf
eng
Massachusetts Institute of Technology
M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.
http://dspace.mit.edu/handle/1721.1/7582
Biology.
Structure determination of bacteriophage [phi]21 N-peptide and boxB RNA complex by NMR spectrocsopy
Thesis
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<Abstract>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.</Abstract>
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