Regulation of gene expression in Escherichia coliilvGMEDA cluster.
Item
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Title
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Regulation of gene expression in Escherichia coliilvGMEDA cluster.
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Identifier
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AAI9304675
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identifier
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9304675
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Creator
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Huang, Fei.
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Contributor
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Adviser: David H. Calhoun
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Date
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1992
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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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Chemistry, Biochemistry
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Abstract
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The ilvGMEDA cluster encodes five gene products needed for the biosynthesis of leucine, isoleucine, and valine in Escherichia coli K-12. While some of the sites of transcription initiation and termination have been identified for the ilvGMEDA cluster, the sizes and distribution of mRNAs present in vivo have not previously been described. In this study, four transcripts have been identified. Two relatively stable transcripts of 4.6 and 1.1 kb correspond to the products of the ilvEDA and ilvE genes, and two relatively unstable transcripts of 6.7 and 3.6 kb correspond to the products of the ilvGMEDA and ilvDA genes. The identity of the transcripts was based on the use of eight probes derived from segments of the ilvGMEDA cluster. In addition, two strains with deletions of ilvG or ilvDA were used, and the expected decrease in transcript size was observed in Northern (RNA) blots. Primer extension using a synthetic oligonucleotide derived from the 5{dollar}\sp\prime{dollar}-end of the ilvD gene and reverse transcriptase generated a 169 nt product corresponding to a 5{dollar}\sp\prime{dollar}-end of transcript within the ilvED intercistronic region, 37 nt from the AUG codon of the ilvD gene. This primer extension product presumably indicates the 5{dollar}\sp\prime{dollar}-end of the ilvDA transcript detected in Northern blots. The stability of the transcripts was monitored following rifampicin addition, and RNase E, RNase P, RNase II and PNPase, but not RNase III, were found to participate in degradation of the ilv transcripts. The ilv transcript levels varied as predicted in response to growth in the presence and absence of the end product amino acids, and in response to the presence of the polar frameshift site in ilvG and regulatory allele ilvA538. In a strain with a rho mutation, the level of 4.6 kb ilvEDA transcript increased, but the 1.1 kb ilvE transcript decreased. This indicated that a Rho dependent termination site may be present in the ilvE-ilvD intercistronic region that causes the release of the 1.1 kb ilvE transcript rather than readthrough to generate the 4.6 kb ilvEDA transcript in wild-type (Rho{dollar}\sp+{dollar}) strain.;It was previously observed that an ilv-related protein, (ORFI), was produced at higher levels in UV irradiated cells infected with {dollar}\lambda{dollar}dilvGMEDA phage with specific ilvG (Val{dollar}\sp{lcub}\rm R{rcub}{dollar}) mutations, compared to phage carrying the wild type ilvG{dollar}\sp+{dollar} (Val{dollar}\sp{lcub}\rm S{rcub}{dollar}) allele. The gene coding for this protein was further localized by analyzing restriction fragment subsets in maxicells to a region between rrnC and ilvGMEDA, approximately 2.1 kb from the ilvGMEDA cluster. In this study the DNA sequence of the 3.5 kb segment between rrnC and ilvGMEDA was determined. Two open reading frames (ORFs) predict proteins of 18,751 (ORFI) and 20,085 (ORFII) daltons, and both ORFs have a strong probability to code for proteins based on codon frequency analysis.
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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.
