Binding of the bacteriophage T4 regA protein to mRNA targets : an initiator AUG is required
Bacteriophage T4 regA protein translationally represses the synthesis of a subset of early phage-induced proteins. The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex. We show here that the protein binds to the translation initi...
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| Veröffentlicht in: | Nucleic acids research 1990-12, Vol.18 (23), p.7083-7092 |
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| creator | UNNITHAN, S GREEN, L MORRISSEY, L BINKLEY, J SINGER, B KARAM, J GOLD, L |
| description | Bacteriophage T4 regA protein translationally represses the synthesis of a subset of early phage-induced proteins. The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex. We show here that the protein binds to the translation initiation sites of other regA-sensitive mRNAs. Analysis of mRNA binding by filtration and nuclease protection assays shows that AUG is necessary but not sufficient for specific binding of regA protein to its mRNA targets. Anticipating the need for large quantities of regA protein for structural studies to further define the regA protein-RNA ligand interaction, we also report cloning the regA gene into a T4 overexpression system. The expression of regA protein in uninfected E. coli is lethal, so in our system regA driven by a strong T7 promoter is sequestered in a T4 phage until 'induction' by phage infection is desired. We have replaced the regA sensitive wild-type ribosome binding site with a strong insensitive ribosome binding site at an optimal distance from the regA initiation codon for maximizing expression. We have obtained large amounts of regA protein. |
| doi_str_mv | 10.1093/nar/18.23.7083 |
| format | Article |
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The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex. We show here that the protein binds to the translation initiation sites of other regA-sensitive mRNAs. Analysis of mRNA binding by filtration and nuclease protection assays shows that AUG is necessary but not sufficient for specific binding of regA protein to its mRNA targets. Anticipating the need for large quantities of regA protein for structural studies to further define the regA protein-RNA ligand interaction, we also report cloning the regA gene into a T4 overexpression system. The expression of regA protein in uninfected E. coli is lethal, so in our system regA driven by a strong T7 promoter is sequestered in a T4 phage until 'induction' by phage infection is desired. We have replaced the regA sensitive wild-type ribosome binding site with a strong insensitive ribosome binding site at an optimal distance from the regA initiation codon for maximizing expression. We have obtained large amounts of regA protein.</description><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/18.23.7083</identifier><identifier>PMID: 2263467</identifier><identifier>CODEN: NARHAD</identifier><language>eng</language><publisher>Oxford: Oxford University Press</publisher><subject>Base Sequence ; Binding Sites ; Binding, Competitive ; Biological and medical sciences ; Cloning, Molecular ; Codon ; Escherichia coli ; Escherichia coli - genetics ; Fundamental and applied biological sciences. Psychology ; Gene Expression Regulation, Viral ; Genes, Viral ; Molecular and cellular biology ; Molecular genetics ; Molecular Sequence Data ; Mutation ; phage T4 ; Protein Biosynthesis ; Ribosomes - metabolism ; RNA, Messenger - genetics ; RNA, Messenger - metabolism ; RNA, Viral - metabolism ; T-Phages - metabolism ; Translation. Translation factors. Protein processing ; Viral Proteins - genetics ; Viral Proteins - isolation & purification ; Viral Proteins - metabolism</subject><ispartof>Nucleic acids research, 1990-12, Vol.18 (23), p.7083-7092</ispartof><rights>1991 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4573-388b4795e979f43140d0caa93780caf2b717c8becb444013f6744e7029081793</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC332773/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC332773/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27922,27923,53789,53791</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=19519745$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2263467$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>UNNITHAN, S</creatorcontrib><creatorcontrib>GREEN, L</creatorcontrib><creatorcontrib>MORRISSEY, L</creatorcontrib><creatorcontrib>BINKLEY, J</creatorcontrib><creatorcontrib>SINGER, B</creatorcontrib><creatorcontrib>KARAM, J</creatorcontrib><creatorcontrib>GOLD, L</creatorcontrib><title>Binding of the bacteriophage T4 regA protein to mRNA targets : an initiator AUG is required</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Res</addtitle><description>Bacteriophage T4 regA protein translationally represses the synthesis of a subset of early phage-induced proteins. The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex. We show here that the protein binds to the translation initiation sites of other regA-sensitive mRNAs. Analysis of mRNA binding by filtration and nuclease protection assays shows that AUG is necessary but not sufficient for specific binding of regA protein to its mRNA targets. Anticipating the need for large quantities of regA protein for structural studies to further define the regA protein-RNA ligand interaction, we also report cloning the regA gene into a T4 overexpression system. The expression of regA protein in uninfected E. coli is lethal, so in our system regA driven by a strong T7 promoter is sequestered in a T4 phage until 'induction' by phage infection is desired. We have replaced the regA sensitive wild-type ribosome binding site with a strong insensitive ribosome binding site at an optimal distance from the regA initiation codon for maximizing expression. We have obtained large amounts of regA protein.</description><subject>Base Sequence</subject><subject>Binding Sites</subject><subject>Binding, Competitive</subject><subject>Biological and medical sciences</subject><subject>Cloning, Molecular</subject><subject>Codon</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Expression Regulation, Viral</subject><subject>Genes, Viral</subject><subject>Molecular and cellular biology</subject><subject>Molecular genetics</subject><subject>Molecular Sequence Data</subject><subject>Mutation</subject><subject>phage T4</subject><subject>Protein Biosynthesis</subject><subject>Ribosomes - metabolism</subject><subject>RNA, Messenger - genetics</subject><subject>RNA, Messenger - metabolism</subject><subject>RNA, Viral - metabolism</subject><subject>T-Phages - metabolism</subject><subject>Translation. Translation factors. Protein processing</subject><subject>Viral Proteins - genetics</subject><subject>Viral Proteins - isolation & purification</subject><subject>Viral Proteins - metabolism</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1990</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc1vEzEQxS0EKqFw5YbkC9w2tT2z_kDiECooSBVIKJw4WF7HuzFK7NR2kPjv2apRgVNPc3i_9zQzj5CXnC05M3CRXLngeilgqZiGR2TBQYoOjRSPyYIB6zvOUD8lz2r9yRhH3uMZORNCAkq1ID_ex7SJaaJ5pG0b6OB8CyXmw9ZNga6RljCt6KHkFmKiLdP9ty8r2lyZQqv0LXWJxhRbdC0Xuvp-RWOdLTfHWMLmOXkyul0NL07znKw_flhffuquv159vlxddx57BR1oPaAyfTDKjAgc2YZ55wwoPc9RDIorr4fgB0RkHEapEINiwjDNlYFz8u4u9nAc9mHjQ2rF7eyhxL0rv2120f6vpLi1U_5lAYRSMPvfnPwl3xxDbXYfqw-7nUshH6vVjGuh5cMgn5-OgOZhUEopeilncHkH-pJrLWG835oze1uvneu1XFsB9rbe2fDq31vv8VOfs_76pLvq3W4sLvlY_6aanhuFPfwBw9isOg</recordid><startdate>19901211</startdate><enddate>19901211</enddate><creator>UNNITHAN, S</creator><creator>GREEN, L</creator><creator>MORRISSEY, L</creator><creator>BINKLEY, J</creator><creator>SINGER, B</creator><creator>KARAM, J</creator><creator>GOLD, L</creator><general>Oxford University Press</general><scope>IQODW</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>7TM</scope><scope>7U9</scope><scope>H94</scope><scope>7T7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19901211</creationdate><title>Binding of the bacteriophage T4 regA protein to mRNA targets : an initiator AUG is required</title><author>UNNITHAN, S ; GREEN, L ; MORRISSEY, L ; BINKLEY, J ; SINGER, B ; KARAM, J ; GOLD, L</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4573-388b4795e979f43140d0caa93780caf2b717c8becb444013f6744e7029081793</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1990</creationdate><topic>Base Sequence</topic><topic>Binding Sites</topic><topic>Binding, Competitive</topic><topic>Biological and medical sciences</topic><topic>Cloning, Molecular</topic><topic>Codon</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Gene Expression Regulation, Viral</topic><topic>Genes, Viral</topic><topic>Molecular and cellular biology</topic><topic>Molecular genetics</topic><topic>Molecular Sequence Data</topic><topic>Mutation</topic><topic>phage T4</topic><topic>Protein Biosynthesis</topic><topic>Ribosomes - metabolism</topic><topic>RNA, Messenger - genetics</topic><topic>RNA, Messenger - metabolism</topic><topic>RNA, Viral - metabolism</topic><topic>T-Phages - metabolism</topic><topic>Translation. Translation factors. Protein processing</topic><topic>Viral Proteins - genetics</topic><topic>Viral Proteins - isolation & purification</topic><topic>Viral Proteins - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>UNNITHAN, S</creatorcontrib><creatorcontrib>GREEN, L</creatorcontrib><creatorcontrib>MORRISSEY, L</creatorcontrib><creatorcontrib>BINKLEY, J</creatorcontrib><creatorcontrib>SINGER, B</creatorcontrib><creatorcontrib>KARAM, J</creatorcontrib><creatorcontrib>GOLD, L</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>UNNITHAN, S</au><au>GREEN, L</au><au>MORRISSEY, L</au><au>BINKLEY, J</au><au>SINGER, B</au><au>KARAM, J</au><au>GOLD, L</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Binding of the bacteriophage T4 regA protein to mRNA targets : an initiator AUG is required</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>1990-12-11</date><risdate>1990</risdate><volume>18</volume><issue>23</issue><spage>7083</spage><epage>7092</epage><pages>7083-7092</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><coden>NARHAD</coden><abstract>Bacteriophage T4 regA protein translationally represses the synthesis of a subset of early phage-induced proteins. The protein binds to the translation initiation site of at least two mRNAs and prevents formation of the initiation complex. We show here that the protein binds to the translation initiation sites of other regA-sensitive mRNAs. Analysis of mRNA binding by filtration and nuclease protection assays shows that AUG is necessary but not sufficient for specific binding of regA protein to its mRNA targets. Anticipating the need for large quantities of regA protein for structural studies to further define the regA protein-RNA ligand interaction, we also report cloning the regA gene into a T4 overexpression system. The expression of regA protein in uninfected E. coli is lethal, so in our system regA driven by a strong T7 promoter is sequestered in a T4 phage until 'induction' by phage infection is desired. 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| ispartof | Nucleic acids research, 1990-12, Vol.18 (23), p.7083-7092 |
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| source | Oxford University Press Journals Digital Archive legacy; MEDLINE; PubMed Central |
| subjects | Base Sequence Binding Sites Binding, Competitive Biological and medical sciences Cloning, Molecular Codon Escherichia coli Escherichia coli - genetics Fundamental and applied biological sciences. Psychology Gene Expression Regulation, Viral Genes, Viral Molecular and cellular biology Molecular genetics Molecular Sequence Data Mutation phage T4 Protein Biosynthesis Ribosomes - metabolism RNA, Messenger - genetics RNA, Messenger - metabolism RNA, Viral - metabolism T-Phages - metabolism Translation. Translation factors. Protein processing Viral Proteins - genetics Viral Proteins - isolation & purification Viral Proteins - metabolism |
| title | Binding of the bacteriophage T4 regA protein to mRNA targets : an initiator AUG is required |
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