S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation
To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient pnp::lacZ fusion reporter strain was used to assess autoregulation by mutant PNPase protein...
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| Veröffentlicht in: | Journal of Bacteriology 2013-05, Vol.195 (9), p.2021-2031 |
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| container_title | Journal of Bacteriology |
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| creator | Wong, Alexander G McBurney, Kristina L Thompson, Katharine J Stickney, Leigh M Mackie, George A |
| description | To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient pnp::lacZ fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using in vitro band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between β-galactosidase levels reflecting autoregulation and apparent KD values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase. |
| doi_str_mv | 10.1128/JB.00062-13 |
| format | Article |
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A convenient pnp::lacZ fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using in vitro band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between β-galactosidase levels reflecting autoregulation and apparent KD values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase.</description><identifier>ISSN: 0021-9193</identifier><identifier>EISSN: 1098-5530</identifier><identifier>EISSN: 1067-8832</identifier><identifier>DOI: 10.1128/JB.00062-13</identifier><identifier>PMID: 23457244</identifier><identifier>CODEN: JOBAAY</identifier><language>eng</language><publisher>United States: American Society for Microbiology</publisher><subject>autoregulation ; Bacteriology ; beta-galactosidase ; Binding sites ; Correlation analysis ; Enzymatic activity ; enzyme activity ; Enzymes ; Escherichia coli - chemistry ; Escherichia coli - enzymology ; Escherichia coli - genetics ; Escherichia coli Proteins - chemistry ; Escherichia coli Proteins - genetics ; Escherichia coli Proteins - metabolism ; Gene Expression Regulation, Enzymologic ; Homeostasis ; Kinetics ; mutants ; Mutation ; phosphorylase ; point mutation ; Polyribonucleotide Nucleotidyltransferase - chemistry ; Polyribonucleotide Nucleotidyltransferase - genetics ; Polyribonucleotide Nucleotidyltransferase - metabolism ; Protein Binding ; Protein Structure, Tertiary ; Proteins ; Ribonucleic acid ; RNA ; RNA, Bacterial - genetics ; RNA, Bacterial - metabolism ; Substrates</subject><ispartof>Journal of Bacteriology, 2013-05, Vol.195 (9), p.2021-2031</ispartof><rights>Copyright American Society for Microbiology May 2013</rights><rights>Copyright © 2013, American Society for Microbiology. All Rights Reserved. 2013 American Society for Microbiology</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c492t-3969a732a0c36cd3b4779c4a048e7e3c69bad62f41be59b7c0e781846f2dd5c43</citedby><cites>FETCH-LOGICAL-c492t-3969a732a0c36cd3b4779c4a048e7e3c69bad62f41be59b7c0e781846f2dd5c43</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624587/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624587/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,315,728,781,785,886,27929,27930,53796,53798</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23457244$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Wong, Alexander G</creatorcontrib><creatorcontrib>McBurney, Kristina L</creatorcontrib><creatorcontrib>Thompson, Katharine J</creatorcontrib><creatorcontrib>Stickney, Leigh M</creatorcontrib><creatorcontrib>Mackie, George A</creatorcontrib><title>S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation</title><title>Journal of Bacteriology</title><addtitle>J Bacteriol</addtitle><description>To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient pnp::lacZ fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using in vitro band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between β-galactosidase levels reflecting autoregulation and apparent KD values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase.</description><subject>autoregulation</subject><subject>Bacteriology</subject><subject>beta-galactosidase</subject><subject>Binding sites</subject><subject>Correlation analysis</subject><subject>Enzymatic activity</subject><subject>enzyme activity</subject><subject>Enzymes</subject><subject>Escherichia coli - chemistry</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli Proteins - chemistry</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Escherichia coli Proteins - metabolism</subject><subject>Gene Expression Regulation, Enzymologic</subject><subject>Homeostasis</subject><subject>Kinetics</subject><subject>mutants</subject><subject>Mutation</subject><subject>phosphorylase</subject><subject>point mutation</subject><subject>Polyribonucleotide Nucleotidyltransferase - chemistry</subject><subject>Polyribonucleotide Nucleotidyltransferase - genetics</subject><subject>Polyribonucleotide Nucleotidyltransferase - metabolism</subject><subject>Protein Binding</subject><subject>Protein Structure, Tertiary</subject><subject>Proteins</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA, Bacterial - genetics</subject><subject>RNA, Bacterial - metabolism</subject><subject>Substrates</subject><issn>0021-9193</issn><issn>1098-5530</issn><issn>1067-8832</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNks1v1DAQxSMEokvhxB2MuCChFH8nvlTabQulVFBRerYcZ7LxKrEXO2m1_z3Z3VIBJ05zmN88vZk3WfaS4CNCaPnhYnGEMZY0J-xRNiNYlbkQDD_OZhhTkiui2EH2LKUVxoRzQZ9mB5RxUVDOZ1l3TZDxNfpyjk5Db5xPKDToKnQbP9oOwuBqQFdtSOs2xE1nEqBTGCD2zgMaWkBnTeOsA28328HvX-do4Xzt_HInOx-HEGE5dmZwwT_PnjSmS_Divh5mNx_Pfpyc55ffPn0-mV_mlis65ExJZQpGDbZM2ppVvCiU5QbzEgpgVqrK1JI2nFQgVFVYDEVJSi4bWtfCcnaYHe9112PVQ23BD9F0eh1db-JGB-P03x3vWr0Mt5pJykVZTALv7gVi-DlCGnTvkoWuMx7CmPR0R8YKKdh_oIxKwhkmYkLf_oOuwhj9dIkdRRUVdGv-_Z6yMaQUoXnwTbDeBq4vFnoX-DQ10a_-XPWB_Z3wBLzZA61btncugjap16tKEyW00nR6kYl5vWcaE7RZRpf0zTWdLG8_RpaSsF_bkLk0</recordid><startdate>20130501</startdate><enddate>20130501</enddate><creator>Wong, Alexander G</creator><creator>McBurney, Kristina L</creator><creator>Thompson, Katharine J</creator><creator>Stickney, Leigh M</creator><creator>Mackie, George A</creator><general>American Society for Microbiology</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20130501</creationdate><title>S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation</title><author>Wong, Alexander G ; McBurney, Kristina L ; Thompson, Katharine J ; Stickney, Leigh M ; Mackie, George A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c492t-3969a732a0c36cd3b4779c4a048e7e3c69bad62f41be59b7c0e781846f2dd5c43</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>autoregulation</topic><topic>Bacteriology</topic><topic>beta-galactosidase</topic><topic>Binding sites</topic><topic>Correlation analysis</topic><topic>Enzymatic activity</topic><topic>enzyme activity</topic><topic>Enzymes</topic><topic>Escherichia coli - chemistry</topic><topic>Escherichia coli - enzymology</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli Proteins - chemistry</topic><topic>Escherichia coli Proteins - genetics</topic><topic>Escherichia coli Proteins - metabolism</topic><topic>Gene Expression Regulation, Enzymologic</topic><topic>Homeostasis</topic><topic>Kinetics</topic><topic>mutants</topic><topic>Mutation</topic><topic>phosphorylase</topic><topic>point mutation</topic><topic>Polyribonucleotide Nucleotidyltransferase - chemistry</topic><topic>Polyribonucleotide Nucleotidyltransferase - genetics</topic><topic>Polyribonucleotide Nucleotidyltransferase - metabolism</topic><topic>Protein Binding</topic><topic>Protein Structure, Tertiary</topic><topic>Proteins</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>RNA, Bacterial - genetics</topic><topic>RNA, Bacterial - metabolism</topic><topic>Substrates</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wong, Alexander G</creatorcontrib><creatorcontrib>McBurney, Kristina L</creatorcontrib><creatorcontrib>Thompson, Katharine J</creatorcontrib><creatorcontrib>Stickney, Leigh M</creatorcontrib><creatorcontrib>Mackie, George A</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of Bacteriology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wong, Alexander G</au><au>McBurney, Kristina L</au><au>Thompson, Katharine J</au><au>Stickney, Leigh M</au><au>Mackie, George A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation</atitle><jtitle>Journal of Bacteriology</jtitle><addtitle>J Bacteriol</addtitle><date>2013-05-01</date><risdate>2013</risdate><volume>195</volume><issue>9</issue><spage>2021</spage><epage>2031</epage><pages>2021-2031</pages><issn>0021-9193</issn><eissn>1098-5530</eissn><eissn>1067-8832</eissn><coden>JOBAAY</coden><abstract>To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient pnp::lacZ fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using in vitro band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between β-galactosidase levels reflecting autoregulation and apparent KD values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase.</abstract><cop>United States</cop><pub>American Society for Microbiology</pub><pmid>23457244</pmid><doi>10.1128/JB.00062-13</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | autoregulation Bacteriology beta-galactosidase Binding sites Correlation analysis Enzymatic activity enzyme activity Enzymes Escherichia coli - chemistry Escherichia coli - enzymology Escherichia coli - genetics Escherichia coli Proteins - chemistry Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism Gene Expression Regulation, Enzymologic Homeostasis Kinetics mutants Mutation phosphorylase point mutation Polyribonucleotide Nucleotidyltransferase - chemistry Polyribonucleotide Nucleotidyltransferase - genetics Polyribonucleotide Nucleotidyltransferase - metabolism Protein Binding Protein Structure, Tertiary Proteins Ribonucleic acid RNA RNA, Bacterial - genetics RNA, Bacterial - metabolism Substrates |
| title | S1 and KH Domains of Polynucleotide Phosphorylase Determine the Efficiency of RNA Binding and Autoregulation |
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