Stability ofmRNAs produced from theilvGMEDA gene cluster of Escherichia coli K-12.
Item
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Title
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Stability ofmRNAs produced from theilvGMEDA gene cluster of Escherichia coli K-12.
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Identifier
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AAI9908388
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identifier
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9908388
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Creator
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Zhou, Chenyi.
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Contributor
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Adviser: David H. Calhoun
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Date
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1998
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular | Biology, Microbiology
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Abstract
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Several lines of evidence revealed the presence of post-transcriptional processing pathways for the ilvGMEDA, ilvEDA, ilvDA, and ilvE transcripts produced by the ilvGMEDA gene cluster of Escherichia coli K-12. First, a mutation in endoribonuclease RNase E (rne-3071{dollar}\sp{lcub}ts{rcub}){dollar} increased the stability of the ilvGMEDA and ilvE transcripts, and a mutation in endoribonuclease RNase P (rnpA49{dollar}\sp{lcub}ts{rcub}){dollar} increased the stability of the ilvEDA and ilvE transcripts. Second, two min after inhibition of transcription initiation by rifampicin addition to a strain with an RNase E mutation, there was a marked decrease in the ilvGMEDA and ilvEDA transcripts that was simultaneous with a marked increase in the ilvE transcript, and there was also a simultaneous increase in partially processed intermediates present just above the ilvE mRNA. Third, the higher levels of the full length ilvGMEDA transcript in the Val{dollar}\sp{lcub}\rm R{rcub}{dollar} compared to Val{dollar}\sp{lcub}\rm S{rcub}{dollar} strains resulted in higher levels of the smaller ilvEDA, ilvE, and ilvDA transcripts in Val{dollar}\sp{lcub}\rm R{rcub}{dollar} strains compared to the Val{dollar}\sp{lcub}\rm S{rcub}{dollar} strain. And fourth, higher levels of the full length ilvGMEDA transcript in the Val{dollar}\sp{lcub}\rm S{rcub}{dollar} strain due to the presence of a Rho mutation also resulted in an increase in the smaller ilvEDA, ilvE, and ilvDA transcripts. These results indicated a processing of ilvGMEDA to ilvEDA, and ilvEDA to ilvE and ilvDA, with some fraction of the ilvEDA transcript formed directly from the constitutive internal ilvEp promoter. In the presence of a rho-115 mutation, about half of the ilvGMEDA transcripts were degraded rather than processed to the smaller mRNAs. This result is related to the observation of Sozhamannan and Stitt (1997) that bulk mRNA levels decreased in the presence of some, but not all, rho mutations. This is the first example of a specific transcript that is less stable in the presence of a rho mutation. The participation of RNase E in the processing of the ilvGMEDA transcript was examined in more detail. RNase E was purified from an E. coli strain containing the gene on a high level expression plasmid. A RNA segment of 1,136 nt containing the ilvE - ilvD intercistronic region was synthesized in vitro. Treatment of this RNA segment with RNase E revealed the presence of several major and minor cleavage sites corresponding to RNase E. This in vitro result supports the interpretation of the in vivo experiments that the ilvE transcript is derived in part from the ilvEDA transcript by RNase E processing.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
