Structural and Functional Properties of Ribosomes Crosslinked with Dimethylsuberimidate
To test whether a 30S ribosomal subunit-formylmethionyl-tRNA-mRNA complex is an obligatory intermediate in protein synthesis, 70S ribosomes from Escherichia coli were crosslinked with the bifunctional imidoester, dimethylsuberimidate. Crosslinked ribosomes contained covalently joined 30S and 50S sub...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1972-12, Vol.69 (12), p.3769-3773 |
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| creator | Slobin, Lawrence I. |
| description | To test whether a 30S ribosomal subunit-formylmethionyl-tRNA-mRNA complex is an obligatory intermediate in protein synthesis, 70S ribosomes from Escherichia coli were crosslinked with the bifunctional imidoester, dimethylsuberimidate. Crosslinked ribosomes contained covalently joined 30S and 50S subunits, as judged by their inability to dissociate at low Mg2+concentrations. Treatment of 70S ribosomes with high salt (1 M NH4Cl), either before or after reaction with the crosslinking reagent, produced two different crosslinked ribosomal particles, one of ``60 S'' and the other ``70 S.'' Preliminary evidence indicates that both particles can bind N-acetylphenylalanyl-tRNA at low Mg2+concentrations and are active for polyphenylalanine syntheses. Crosslinked ribosomes were functional when tested with poly(U) as an mRNA in systems requiring initiation factors and N-acetylphenylalanyl-tRNA for activity. Under optimal crosslinking conditions, they retained 80% of the activity of unmodified ribosomes for polyphenylalanine synthesis. Despite the maintenance of these functional capacities, such ribosomes had a sharply reduced ability to bind fMet-tRNA and were completely inactive in protein synthesis with bacteriophage f2 RNA as a messenger. We conclude that 70S ribosomes must dissociate into subunits to initiate protein synthesis with natural mRNAs. |
| doi_str_mv | 10.1073/pnas.69.12.3769 |
| format | Article |
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Crosslinked ribosomes contained covalently joined 30S and 50S subunits, as judged by their inability to dissociate at low Mg2+concentrations. Treatment of 70S ribosomes with high salt (1 M NH4Cl), either before or after reaction with the crosslinking reagent, produced two different crosslinked ribosomal particles, one of ``60 S'' and the other ``70 S.'' Preliminary evidence indicates that both particles can bind N-acetylphenylalanyl-tRNA at low Mg2+concentrations and are active for polyphenylalanine syntheses. Crosslinked ribosomes were functional when tested with poly(U) as an mRNA in systems requiring initiation factors and N-acetylphenylalanyl-tRNA for activity. Under optimal crosslinking conditions, they retained 80% of the activity of unmodified ribosomes for polyphenylalanine synthesis. Despite the maintenance of these functional capacities, such ribosomes had a sharply reduced ability to bind fMet-tRNA and were completely inactive in protein synthesis with bacteriophage f2 RNA as a messenger. We conclude that 70S ribosomes must dissociate into subunits to initiate protein synthesis with natural mRNAs.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.69.12.3769</identifier><identifier>PMID: 4566460</identifier><language>eng</language><publisher>United States: National Academy of Sciences of the United States of America</publisher><subject>Bacterial Proteins - biosynthesis ; Bacteriophages ; Biological Sciences: Biochemistry ; Carbon Isotopes ; Centrifugation ; Centrifugation, Density Gradient ; Coliphages ; Crosslinking ; Dicarboxylic Acids - metabolism ; Escherichia coli - metabolism ; Imides - metabolism ; Messenger RNA ; Methionine ; Molecular Weight ; Peptide Chain Initiation, Translational ; Peptide Initiation Factors ; Phenylalanine - biosynthesis ; Poly U - metabolism ; Protein synthesis ; Puromycin - pharmacology ; Ribosomes ; Ribosomes - analysis ; Ribosomes - drug effects ; Ribosomes - metabolism ; RNA ; RNA, Ribosomal - isolation & purification ; RNA, Ribosomal - metabolism ; RNA, Transfer - metabolism ; RNA, Viral - metabolism ; Transfer RNA ; Valine - metabolism ; Washing</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1972-12, Vol.69 (12), p.3769-3773</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c461t-70b1ba6bc0823e203f41e88ab1626cf5ad8eaa3076234604b15532bc04e877b73</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/69/12.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/62083$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/62083$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,725,778,782,801,883,27907,27908,53774,53776,58000,58233</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/4566460$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Slobin, Lawrence I.</creatorcontrib><title>Structural and Functional Properties of Ribosomes Crosslinked with Dimethylsuberimidate</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>To test whether a 30S ribosomal subunit-formylmethionyl-tRNA-mRNA complex is an obligatory intermediate in protein synthesis, 70S ribosomes from Escherichia coli were crosslinked with the bifunctional imidoester, dimethylsuberimidate. Crosslinked ribosomes contained covalently joined 30S and 50S subunits, as judged by their inability to dissociate at low Mg2+concentrations. Treatment of 70S ribosomes with high salt (1 M NH4Cl), either before or after reaction with the crosslinking reagent, produced two different crosslinked ribosomal particles, one of ``60 S'' and the other ``70 S.'' Preliminary evidence indicates that both particles can bind N-acetylphenylalanyl-tRNA at low Mg2+concentrations and are active for polyphenylalanine syntheses. Crosslinked ribosomes were functional when tested with poly(U) as an mRNA in systems requiring initiation factors and N-acetylphenylalanyl-tRNA for activity. Under optimal crosslinking conditions, they retained 80% of the activity of unmodified ribosomes for polyphenylalanine synthesis. Despite the maintenance of these functional capacities, such ribosomes had a sharply reduced ability to bind fMet-tRNA and were completely inactive in protein synthesis with bacteriophage f2 RNA as a messenger. We conclude that 70S ribosomes must dissociate into subunits to initiate protein synthesis with natural mRNAs.</description><subject>Bacterial Proteins - biosynthesis</subject><subject>Bacteriophages</subject><subject>Biological Sciences: Biochemistry</subject><subject>Carbon Isotopes</subject><subject>Centrifugation</subject><subject>Centrifugation, Density Gradient</subject><subject>Coliphages</subject><subject>Crosslinking</subject><subject>Dicarboxylic Acids - metabolism</subject><subject>Escherichia coli - metabolism</subject><subject>Imides - metabolism</subject><subject>Messenger RNA</subject><subject>Methionine</subject><subject>Molecular Weight</subject><subject>Peptide Chain Initiation, Translational</subject><subject>Peptide Initiation Factors</subject><subject>Phenylalanine - biosynthesis</subject><subject>Poly U - metabolism</subject><subject>Protein synthesis</subject><subject>Puromycin - pharmacology</subject><subject>Ribosomes</subject><subject>Ribosomes - analysis</subject><subject>Ribosomes - drug effects</subject><subject>Ribosomes - metabolism</subject><subject>RNA</subject><subject>RNA, Ribosomal - isolation & purification</subject><subject>RNA, Ribosomal - metabolism</subject><subject>RNA, Transfer - metabolism</subject><subject>RNA, Viral - metabolism</subject><subject>Transfer RNA</subject><subject>Valine - metabolism</subject><subject>Washing</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1972</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kctv1DAQxi1E1S6FMxISKCc4ZetXbOfAodo-pUogHurRspNJ1yWJF9sp9L_H0W5buHCyRt_vG38zg9BrgpcES3a0GU1cinpJ6JJJUT9DC4JrUgpe4-dogTGVpeKUH6AXMd5ijOtK4X20zyshuMALdP01halJUzB9Yca2OJvGJjk_5vJz8BsIyUEsfFd8cdZHP-RiFXyMvRt_QFv8cmldnLgB0vq-j5OF4AbXmgQv0V5n-givdu8h-n52-m11UV59Or9cHV-VDRcklRJbYo2wDVaUAcWs4wSUMpYIKpquMq0CYxiWgrKcl1tSVYxmnIOS0kp2iD5u-24mO0DbwJjyKHqTc5hwr71x-l9ldGt94-80U7USKvvf7_zB_5wgJj242EDfmxH8FLUiolaSz-DRFmzm8QN0j38QrOdT6PkUWtSaUD2fIjve_h3tkd_tPusfdvpsfFCfGuhu6vsEv1Mm3_2XzMCbLXAbkw9PBMWKsT-h4aja</recordid><startdate>19721201</startdate><enddate>19721201</enddate><creator>Slobin, Lawrence I.</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad Sciences</general><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>7X8</scope><scope>5PM</scope></search><sort><creationdate>19721201</creationdate><title>Structural and Functional Properties of Ribosomes Crosslinked with Dimethylsuberimidate</title><author>Slobin, Lawrence I.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c461t-70b1ba6bc0823e203f41e88ab1626cf5ad8eaa3076234604b15532bc04e877b73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1972</creationdate><topic>Bacterial Proteins - biosynthesis</topic><topic>Bacteriophages</topic><topic>Biological Sciences: Biochemistry</topic><topic>Carbon Isotopes</topic><topic>Centrifugation</topic><topic>Centrifugation, Density Gradient</topic><topic>Coliphages</topic><topic>Crosslinking</topic><topic>Dicarboxylic Acids - metabolism</topic><topic>Escherichia coli - metabolism</topic><topic>Imides - metabolism</topic><topic>Messenger RNA</topic><topic>Methionine</topic><topic>Molecular Weight</topic><topic>Peptide Chain Initiation, Translational</topic><topic>Peptide Initiation Factors</topic><topic>Phenylalanine - biosynthesis</topic><topic>Poly U - metabolism</topic><topic>Protein synthesis</topic><topic>Puromycin - pharmacology</topic><topic>Ribosomes</topic><topic>Ribosomes - analysis</topic><topic>Ribosomes - drug effects</topic><topic>Ribosomes - metabolism</topic><topic>RNA</topic><topic>RNA, Ribosomal - isolation & purification</topic><topic>RNA, Ribosomal - metabolism</topic><topic>RNA, Transfer - metabolism</topic><topic>RNA, Viral - metabolism</topic><topic>Transfer RNA</topic><topic>Valine - metabolism</topic><topic>Washing</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Slobin, Lawrence I.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Slobin, Lawrence I.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Structural and Functional Properties of Ribosomes Crosslinked with Dimethylsuberimidate</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1972-12-01</date><risdate>1972</risdate><volume>69</volume><issue>12</issue><spage>3769</spage><epage>3773</epage><pages>3769-3773</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>To test whether a 30S ribosomal subunit-formylmethionyl-tRNA-mRNA complex is an obligatory intermediate in protein synthesis, 70S ribosomes from Escherichia coli were crosslinked with the bifunctional imidoester, dimethylsuberimidate. Crosslinked ribosomes contained covalently joined 30S and 50S subunits, as judged by their inability to dissociate at low Mg2+concentrations. Treatment of 70S ribosomes with high salt (1 M NH4Cl), either before or after reaction with the crosslinking reagent, produced two different crosslinked ribosomal particles, one of ``60 S'' and the other ``70 S.'' Preliminary evidence indicates that both particles can bind N-acetylphenylalanyl-tRNA at low Mg2+concentrations and are active for polyphenylalanine syntheses. Crosslinked ribosomes were functional when tested with poly(U) as an mRNA in systems requiring initiation factors and N-acetylphenylalanyl-tRNA for activity. Under optimal crosslinking conditions, they retained 80% of the activity of unmodified ribosomes for polyphenylalanine synthesis. Despite the maintenance of these functional capacities, such ribosomes had a sharply reduced ability to bind fMet-tRNA and were completely inactive in protein synthesis with bacteriophage f2 RNA as a messenger. We conclude that 70S ribosomes must dissociate into subunits to initiate protein synthesis with natural mRNAs.</abstract><cop>United States</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>4566460</pmid><doi>10.1073/pnas.69.12.3769</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Bacterial Proteins - biosynthesis Bacteriophages Biological Sciences: Biochemistry Carbon Isotopes Centrifugation Centrifugation, Density Gradient Coliphages Crosslinking Dicarboxylic Acids - metabolism Escherichia coli - metabolism Imides - metabolism Messenger RNA Methionine Molecular Weight Peptide Chain Initiation, Translational Peptide Initiation Factors Phenylalanine - biosynthesis Poly U - metabolism Protein synthesis Puromycin - pharmacology Ribosomes Ribosomes - analysis Ribosomes - drug effects Ribosomes - metabolism RNA RNA, Ribosomal - isolation & purification RNA, Ribosomal - metabolism RNA, Transfer - metabolism RNA, Viral - metabolism Transfer RNA Valine - metabolism Washing |
| title | Structural and Functional Properties of Ribosomes Crosslinked with Dimethylsuberimidate |
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