Post-transcriptional regulation of the steady-state levels of mitochondrial tRNAs in HeLa cells
In human mitochondrial DNA (mtDNA), the tRNA genes are located in three different transcription units that are transcribed at three different rates. To analyze the regulation of tRNA formation by the three transcription units, we have examined the steady-state levels and metabolic properties of the...
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| Veröffentlicht in: | The Journal of biological chemistry 1993-05, Vol.268 (14), p.10228-10237 |
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| description | In human mitochondrial DNA (mtDNA), the tRNA genes are located in three different transcription units that are transcribed at three different rates. To analyze the regulation of tRNA formation by the three transcription units, we have examined the steady-state levels and metabolic properties of the tRNAs of HeLa cell mitochondria. DNA excess hybridization experiments utilizing separated strands of mtDNA and purified tRNA samples from exponential cells long term labeled with [32P]orthophosphate have revealed a steady-state level of 6 x 10(5) tRNA molecules/cell, with three-fourths being encoded in the H-strand and one-fourth in the L-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two-dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNA(Phe) and tRNA(Val) are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs isolated from cells labeled with very short pulses of [5-3H]uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady-state amounts. Therefore, the approximately 25-fold higher rate of transcription of the tRNA(Phe) and tRNA(Val) genes relative to the other H-strand tRNA genes and the 10-16-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes are not reflected in the steady-state levels or the rates of formation of the corresponding tRNAs. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters. |
| doi_str_mv | 10.1016/S0021-9258(18)82194-9 |
| format | Article |
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To analyze the regulation of tRNA formation by the three transcription units, we have examined the steady-state levels and metabolic properties of the tRNAs of HeLa cell mitochondria. DNA excess hybridization experiments utilizing separated strands of mtDNA and purified tRNA samples from exponential cells long term labeled with [32P]orthophosphate have revealed a steady-state level of 6 x 10(5) tRNA molecules/cell, with three-fourths being encoded in the H-strand and one-fourth in the L-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two-dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNA(Phe) and tRNA(Val) are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs isolated from cells labeled with very short pulses of [5-3H]uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady-state amounts. Therefore, the approximately 25-fold higher rate of transcription of the tRNA(Phe) and tRNA(Val) genes relative to the other H-strand tRNA genes and the 10-16-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes are not reflected in the steady-state levels or the rates of formation of the corresponding tRNAs. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters.</description><identifier>ISSN: 0021-9258</identifier><identifier>EISSN: 1083-351X</identifier><identifier>DOI: 10.1016/S0021-9258(18)82194-9</identifier><identifier>PMID: 7683672</identifier><identifier>CODEN: JBCHA3</identifier><language>eng</language><publisher>Bethesda, MD: Elsevier Inc</publisher><subject>Analytical, structural and metabolic biochemistry ; Biological and medical sciences ; Cloning, Molecular ; Codon - metabolism ; DNA, Mitochondrial - genetics ; DNA, Mitochondrial - isolation & purification ; Electrophoresis, Gel, Two-Dimensional ; Electrophoresis, Polyacrylamide Gel ; Fundamental and applied biological sciences. Psychology ; HeLa Cells ; Humans ; Kinetics ; Methionine - metabolism ; Mitochondria - metabolism ; Nucleic Acid Hybridization ; Nucleic acids ; Protein Biosynthesis ; Restriction Mapping ; Ribosomes - metabolism ; RNA - genetics ; RNA - isolation & purification ; RNA - metabolism ; RNA Processing, Post-Transcriptional ; RNA, Messenger - metabolism ; RNA, Mitochondrial ; Rna, ribonucleoproteins ; RNA, Transfer - genetics ; RNA, Transfer - isolation & purification ; RNA, Transfer - metabolism ; Time Factors</subject><ispartof>The Journal of biological chemistry, 1993-05, Vol.268 (14), p.10228-10237</ispartof><rights>1993 © 1993 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.</rights><rights>1993 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4779-f14f21dc0aa55d65ced8c2b6c1945452e11866bcc05c99443aef6a8f135a18853</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27923,27924</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=4771662$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/7683672$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>King, M.P.</creatorcontrib><creatorcontrib>Attardi, G.</creatorcontrib><title>Post-transcriptional regulation of the steady-state levels of mitochondrial tRNAs in HeLa cells</title><title>The Journal of biological chemistry</title><addtitle>J Biol Chem</addtitle><description>In human mitochondrial DNA (mtDNA), the tRNA genes are located in three different transcription units that are transcribed at three different rates. To analyze the regulation of tRNA formation by the three transcription units, we have examined the steady-state levels and metabolic properties of the tRNAs of HeLa cell mitochondria. DNA excess hybridization experiments utilizing separated strands of mtDNA and purified tRNA samples from exponential cells long term labeled with [32P]orthophosphate have revealed a steady-state level of 6 x 10(5) tRNA molecules/cell, with three-fourths being encoded in the H-strand and one-fourth in the L-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two-dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNA(Phe) and tRNA(Val) are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs isolated from cells labeled with very short pulses of [5-3H]uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady-state amounts. Therefore, the approximately 25-fold higher rate of transcription of the tRNA(Phe) and tRNA(Val) genes relative to the other H-strand tRNA genes and the 10-16-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes are not reflected in the steady-state levels or the rates of formation of the corresponding tRNAs. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Biological and medical sciences</subject><subject>Cloning, Molecular</subject><subject>Codon - metabolism</subject><subject>DNA, Mitochondrial - genetics</subject><subject>DNA, Mitochondrial - isolation & purification</subject><subject>Electrophoresis, Gel, Two-Dimensional</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>HeLa Cells</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Methionine - metabolism</subject><subject>Mitochondria - metabolism</subject><subject>Nucleic Acid Hybridization</subject><subject>Nucleic acids</subject><subject>Protein Biosynthesis</subject><subject>Restriction Mapping</subject><subject>Ribosomes - metabolism</subject><subject>RNA - genetics</subject><subject>RNA - isolation & purification</subject><subject>RNA - metabolism</subject><subject>RNA Processing, Post-Transcriptional</subject><subject>RNA, Messenger - metabolism</subject><subject>RNA, Mitochondrial</subject><subject>Rna, ribonucleoproteins</subject><subject>RNA, Transfer - genetics</subject><subject>RNA, Transfer - isolation & purification</subject><subject>RNA, Transfer - metabolism</subject><subject>Time Factors</subject><issn>0021-9258</issn><issn>1083-351X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU9r3DAQxUVpSbdpP0LAh1Lag1uNLMnSqYTQJoElKf0DvQmtPM6q2NZG0ibk21fOLktu0UWI-b2Z0XuEnAD9DBTkl1-UMqg1E-ojqE-Kgea1fkEWQFVTNwL-viSLA_KavEnpHy2HazgiR61UjWzZgpgfIeU6RzslF_0m-zDZoYp4sx3s_KhCX-U1Vimj7R7qlG3GasA7HNJcGn0Obh2mLvoiyz-vTlPlp-oCl7ZyOAzpLXnV2yHhu_19TP58__b77KJeXp9fnp0ua8fbVtc98J5B56i1QnRSOOyUYyvpyq8EFwwBlJQr56hwWnPeWOylVT00woJSojkmH3Z9NzHcbjFlM_o0b2AnDNtkWtGWDvx5EKTQSkJbQLEDXQwpRezNJvrRxgcD1MwJmMcEzGyvAWUeEzC66E72A7arEbuDam95qb_f121yduiL9c6nA1bsACmfYGt_s773Ec3KF6txNEyWebyswJgq2NcdVgLBO4_RJOdxKv4VicumC_6Zff8DskKuOQ</recordid><startdate>19930515</startdate><enddate>19930515</enddate><creator>King, M.P.</creator><creator>Attardi, G.</creator><general>Elsevier Inc</general><general>American Society for Biochemistry and Molecular Biology</general><scope>6I.</scope><scope>AAFTH</scope><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>7X8</scope></search><sort><creationdate>19930515</creationdate><title>Post-transcriptional regulation of the steady-state levels of mitochondrial tRNAs in HeLa cells</title><author>King, M.P. ; Attardi, G.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4779-f14f21dc0aa55d65ced8c2b6c1945452e11866bcc05c99443aef6a8f135a18853</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Biological and medical sciences</topic><topic>Cloning, Molecular</topic><topic>Codon - metabolism</topic><topic>DNA, Mitochondrial - genetics</topic><topic>DNA, Mitochondrial - isolation & purification</topic><topic>Electrophoresis, Gel, Two-Dimensional</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>HeLa Cells</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Methionine - metabolism</topic><topic>Mitochondria - metabolism</topic><topic>Nucleic Acid Hybridization</topic><topic>Nucleic acids</topic><topic>Protein Biosynthesis</topic><topic>Restriction Mapping</topic><topic>Ribosomes - metabolism</topic><topic>RNA - genetics</topic><topic>RNA - isolation & purification</topic><topic>RNA - metabolism</topic><topic>RNA Processing, Post-Transcriptional</topic><topic>RNA, Messenger - metabolism</topic><topic>RNA, Mitochondrial</topic><topic>Rna, ribonucleoproteins</topic><topic>RNA, Transfer - genetics</topic><topic>RNA, Transfer - isolation & purification</topic><topic>RNA, Transfer - metabolism</topic><topic>Time Factors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>King, M.P.</creatorcontrib><creatorcontrib>Attardi, G.</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><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>MEDLINE - Academic</collection><jtitle>The Journal of biological chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>King, M.P.</au><au>Attardi, G.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Post-transcriptional regulation of the steady-state levels of mitochondrial tRNAs in HeLa cells</atitle><jtitle>The Journal of biological chemistry</jtitle><addtitle>J Biol Chem</addtitle><date>1993-05-15</date><risdate>1993</risdate><volume>268</volume><issue>14</issue><spage>10228</spage><epage>10237</epage><pages>10228-10237</pages><issn>0021-9258</issn><eissn>1083-351X</eissn><coden>JBCHA3</coden><abstract>In human mitochondrial DNA (mtDNA), the tRNA genes are located in three different transcription units that are transcribed at three different rates. To analyze the regulation of tRNA formation by the three transcription units, we have examined the steady-state levels and metabolic properties of the tRNAs of HeLa cell mitochondria. DNA excess hybridization experiments utilizing separated strands of mtDNA and purified tRNA samples from exponential cells long term labeled with [32P]orthophosphate have revealed a steady-state level of 6 x 10(5) tRNA molecules/cell, with three-fourths being encoded in the H-strand and one-fourth in the L-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two-dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNA(Phe) and tRNA(Val) are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs isolated from cells labeled with very short pulses of [5-3H]uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady-state amounts. Therefore, the approximately 25-fold higher rate of transcription of the tRNA(Phe) and tRNA(Val) genes relative to the other H-strand tRNA genes and the 10-16-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes are not reflected in the steady-state levels or the rates of formation of the corresponding tRNAs. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters.</abstract><cop>Bethesda, MD</cop><pub>Elsevier Inc</pub><pmid>7683672</pmid><doi>10.1016/S0021-9258(18)82194-9</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | The Journal of biological chemistry, 1993-05, Vol.268 (14), p.10228-10237 |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Analytical, structural and metabolic biochemistry Biological and medical sciences Cloning, Molecular Codon - metabolism DNA, Mitochondrial - genetics DNA, Mitochondrial - isolation & purification Electrophoresis, Gel, Two-Dimensional Electrophoresis, Polyacrylamide Gel Fundamental and applied biological sciences. Psychology HeLa Cells Humans Kinetics Methionine - metabolism Mitochondria - metabolism Nucleic Acid Hybridization Nucleic acids Protein Biosynthesis Restriction Mapping Ribosomes - metabolism RNA - genetics RNA - isolation & purification RNA - metabolism RNA Processing, Post-Transcriptional RNA, Messenger - metabolism RNA, Mitochondrial Rna, ribonucleoproteins RNA, Transfer - genetics RNA, Transfer - isolation & purification RNA, Transfer - metabolism Time Factors |
| title | Post-transcriptional regulation of the steady-state levels of mitochondrial tRNAs in HeLa cells |
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