The mechanism ofermCmRNA degradation in Bacillus subtilis.
Item
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Title
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The mechanism ofermCmRNA degradation in Bacillus subtilis.
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Identifier
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AAI9207142
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identifier
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9207142
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Creator
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Zen, Kuo Huei.
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Contributor
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Adviser: David H. Bechhofer
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Date
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1991
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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
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Abstract
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The ermC gene encodes an rRNA methyltransferase and confers erythromycin resistance on the host by reducing the affinity of ribosome for erythromycin. A previous study had shown that ermC mRNA is stabilized in the presence of erythromycin, an inducer of ermC gene expression. The induced stability of ermC mRNA requires a ribosome stalled in the ermC leader peptide sequence. Our working hypothesis was that the 5{dollar}\sp\prime{dollar} end of ermC mRNA is the target of decay and ribosome stalling in the leader peptide sequence protects ermC mRNA from ribonucleolytic decay in the 5{dollar}\sp\prime{dollar}-to-3{dollar}\sp\prime{dollar} direction.;In order to test this hypothesis, several insertion mutations of the ermC leader region were constructed and used to examine how ribosome stalling affects ermC mRNA stability. Using constructs in which the ribosome stall site was internal rather than at the 5{dollar}\sp\prime{dollar} end of the message, it was shown that ribosome stalling provides stability to sequences downstream but not upstream of the ribosome stall site. In a construct that contained a ribosome stall site at the 5{dollar}\sp\prime{dollar} end, in addition to an internal ribosome stall site, the full-length RNA was stabilized in the presence of erythromycin. These results strongly suggest that ermC mRNA is degraded either by a 5{dollar}\sp\prime{dollar}-to-3{dollar}\sp\prime{dollar} exoribonuclease or by an endoribonuclease that binds to 5{dollar}\sp\prime{dollar} end and make endoribonucleolytic cleavages as it progresses in the 5{dollar}\sp\prime{dollar}-to-3{dollar}\sp\prime{dollar} direction, and that a stalled ribosome at the 5{dollar}\sp\prime{dollar} end protects against this type of decay.;To provide biochemical evidence for this conclusion, B. subtilis extracts were prepared, and RNase substrates were synthesized that could be used to identify a 5{dollar}\sp\prime{dollar} end-requiring RNase activity in B. subtilis extracts. First, it was shown that ermC mRNA synthesized by in vitro transcription is degraded in B. subtilis extracts. Second, an RNA-DNA joint molecule was synthesized that contained an RNA moiety in its 5{dollar}\sp\prime{dollar} and a ssDNA moiety in its 3{dollar}\sp\prime{dollar} region. This joint molecule is degraded to a ssDNA molecule under the condition that degradation of DNA was inhibited, indicating that the RNA moiety of the joint molecule is degraded either by a 5{dollar}\sp\prime{dollar}-to-3{dollar}\sp\prime{dollar} exoribonclease or by an endoribonuclease activity in B. subtilis extracts. To distinguish between these two possibilities, a circular RNA molecule was constructed. This circular RNA was not degraded in B. subtilis extracts although the linear RNA containing the same sequence as the circular RNA was degraded. Taken together, these results suggest the existence of a 5{dollar}\sp\prime{dollar}-to-3{dollar}\sp\prime{dollar} exoribonuclease in B. subtilis.
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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.
