{"id":{"repo_id":"uiuc","oai_identifier":"oai:www.ideals.illinois.edu:2142/70577"},"canonical_url":"https://search.dev.ndltd.org/etd/uiuc/oai:www.ideals.illinois.edu:2142/70577","repository":{"repo_id":"uiuc","name":"University of Illinois - Urbana-Champaign","base_url":"https://www.ideals.illinois.edu/oai-pmh"},"display":{"title":"Identification of Protein-Phosphate Contacts in the R17 Coat Protein Binding Site","abstract":"Small RNAs of defined length and sequence can be synthesized enzymatically using T7 RNA polymerase and synthetic DNA templates which contain the T7 promoter. The templates are base paired from $-17$ to +1 and contain a long 5$\\sp\\prime$ overhang. The products of the transcription reactions have the length and sequence expected for a runoff transcript but may have one or more extra nucleotide at the 3$\\sp\\prime$ terminus. In addition to the full length products, large amounts of smaller products in the range from 2 to 6 nucleotides are produced. These products appear to be the result of abortive initiation events. Variants in the promoter region from +1 to +6 have been made to increase the variety of RNAs which can be synthesized and their efficiency as templates determined. Finally, the reaction conditions have been optimized to allow for the synthesis of milligram amounts of virtually any RNA from 15 to 35 nucleotides in length.","abstract_html":"Small RNAs of defined length and sequence can be synthesized enzymatically using T7 RNA polymerase and synthetic DNA templates which contain the T7 promoter. The templates are base paired from $-17$ to +1 and contain a long 5$\\sp\\prime$ overhang. The products of the transcription reactions have the length and sequence expected for a runoff transcript but may have one or more extra nucleotide at the 3$\\sp\\prime$ terminus. In addition to the full length products, large amounts of smaller products in the range from 2 to 6 nucleotides are produced. These products appear to be the result of abortive initiation events. Variants in the promoter region from +1 to +6 have been made to increase the variety of RNAs which can be synthesized and their efficiency as templates determined. Finally, the reaction conditions have been optimized to allow for the synthesis of milligram amounts of virtually any RNA from 15 to 35 nucleotides in length.","abstract_has_math":true,"creators":["Milligan, John Fairbanks"],"institution":"University of Illinois at Urbana-Champaign","degree_name":"Ph.D.","degree_level":"Dissertation","degree_discipline":"Biochemistry","degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2014,"date_issued":"2014-12-15T23:44:12Z","date_published":"2014-12-15T23:44:12Z","updated_at":"2026-07-22T22:26:03Z","subjects":["Chemistry, Biochemistry"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["(UMI)AAI8908781"],"render_values":[{"text":"(UMI)AAI8908781","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/2142/70577","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Milligan, John Fairbanks"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2014-12-15T23:44:12Z","10000-01-01","1988"]},{"key":"dc:type","label":"Dc Type","values":["text"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["University of Illinois at Urbana-Champaign"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Chemistry, Biochemistry"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/2142/70577","(UMI)AAI8908781"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Small RNAs of defined length and sequence can be synthesized enzymatically using T7 RNA polymerase and synthetic DNA templates which contain the T7 promoter. The templates are base paired from $-17$ to +1 and contain a long 5$\\sp\\prime$ overhang. The products of the transcription reactions have the length and sequence expected for a runoff transcript but may have one or more extra nucleotide at the 3$\\sp\\prime$ terminus. In addition to the full length products, large amounts of smaller products in the range from 2 to 6 nucleotides are produced. These products appear to be the result of abortive initiation events. Variants in the promoter region from +1 to +6 have been made to increase the variety of RNAs which can be synthesized and their efficiency as templates determined. Finally, the reaction conditions have been optimized to allow for the synthesis of milligram amounts of virtually any RNA from 15 to 35 nucleotides in length.","The binding of the bacteriophage R17 coat protein to its RNA binding site is an example of a specific RNA-protein interaction. Extensive analysis has revealed that the binding is dependent upon a unique hairpin structure which contains four essential single stranded nucleotides. Additional specificity is due to 4 or 5 ionic contacts between the protein and the RNA. Transcription of synthetic DNA with T7 RNA polymerase, using one of the nucleoside 5$\\sp\\prime$-O-(1-thiotriphosphate) (NTP($\\alpha$S)), allows the synthesis of RNAs specifically substituted with thiophosphates. Eleven sequence variants of the R17 coat protein binding site, substituted with NTP($\\alpha$S)s, were tested for coat protein binding to deduce the positions of thiophosphates which alter the binding affinity. Of the 21 phosphates in the molecule, two positions decrease the Ka 3-fold, one position decreases the Ka 10-fold, and one position increases the Ka 10-fold. Substitution at any of the other 17 positions does not effect the Ka. The four positions which alter the Ka are located in a uniquely structured region of the RNA, and it is postulated that these thiophosphates effect binding because they are directly contacted by the coat protein.","Made available in DSpace on 2014-12-15T23:44:12Z (GMT). No. of bitstreams: 1 8908781.pdf: 4774115 bytes, checksum: 3b5fc0a712e1d21d77fb3f3d119c1d40 (MD5) Previous issue date: 1988","Embargo set by: Seth Robbins for item 70743 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","U of I Only","137 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988."]},{"key":"dc:title","label":"Title","values":["Identification of Protein-Phosphate Contacts in the R17 Coat Protein Binding Site"]}]}],"canonical_facts":{"dc:creator":["Milligan, John Fairbanks"],"dc:date":["2014-12-15T23:44:12Z","10000-01-01","1988"],"dc:description":["Small RNAs of defined length and sequence can be synthesized enzymatically using T7 RNA polymerase and synthetic DNA templates which contain the T7 promoter. The templates are base paired from $-17$ to +1 and contain a long 5$\\sp\\prime$ overhang. The products of the transcription reactions have the length and sequence expected for a runoff transcript but may have one or more extra nucleotide at the 3$\\sp\\prime$ terminus. In addition to the full length products, large amounts of smaller products in the range from 2 to 6 nucleotides are produced. These products appear to be the result of abortive initiation events. Variants in the promoter region from +1 to +6 have been made to increase the variety of RNAs which can be synthesized and their efficiency as templates determined. Finally, the reaction conditions have been optimized to allow for the synthesis of milligram amounts of virtually any RNA from 15 to 35 nucleotides in length.","The binding of the bacteriophage R17 coat protein to its RNA binding site is an example of a specific RNA-protein interaction. Extensive analysis has revealed that the binding is dependent upon a unique hairpin structure which contains four essential single stranded nucleotides. Additional specificity is due to 4 or 5 ionic contacts between the protein and the RNA. Transcription of synthetic DNA with T7 RNA polymerase, using one of the nucleoside 5$\\sp\\prime$-O-(1-thiotriphosphate) (NTP($\\alpha$S)), allows the synthesis of RNAs specifically substituted with thiophosphates. Eleven sequence variants of the R17 coat protein binding site, substituted with NTP($\\alpha$S)s, were tested for coat protein binding to deduce the positions of thiophosphates which alter the binding affinity. Of the 21 phosphates in the molecule, two positions decrease the Ka 3-fold, one position decreases the Ka 10-fold, and one position increases the Ka 10-fold. Substitution at any of the other 17 positions does not effect the Ka. The four positions which alter the Ka are located in a uniquely structured region of the RNA, and it is postulated that these thiophosphates effect binding because they are directly contacted by the coat protein.","Made available in DSpace on 2014-12-15T23:44:12Z (GMT). No. of bitstreams: 1 8908781.pdf: 4774115 bytes, checksum: 3b5fc0a712e1d21d77fb3f3d119c1d40 (MD5) Previous issue date: 1988","Embargo set by: Seth Robbins for item 70743 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs","U of I Only","137 p.","Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988."],"dc:identifier":["http://hdl.handle.net/2142/70577","(UMI)AAI8908781"],"dc:subject":["Chemistry, Biochemistry"],"dc:title":["Identification of Protein-Phosphate Contacts in the R17 Coat Protein Binding Site"],"dc:type":["text"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_level":["Dissertation"],"thesis:degree_name":["Ph.D."],"thesis:institution_name":["University of Illinois at Urbana-Champaign"]},"updated_at":"2026-07-22T22:26:03Z"}