Title page for ETD etd-43431593973981


Type of Document Dissertation
Author Solow, Steven P.
Author's Email Address solowsp@vt.edu
URN etd-43431593973981
Title Characterization of A transcriptional Attenuator in The rpmf-Plsx-Fab Operon of Escherichia coli K-12
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
No Advisors Found
Keywords
  • transcription
  • s.i.t.k.g
  • phospholipids
  • fatty acids
  • attenuator
  • Escherichia coli
Date of Defense 1997-07-04
Availability unrestricted
Abstract

Fatty acids are an essential component of the

phospholipids of the inner and outer membranes of

Escherichia coli. The synthesis of both fatty acids and

phospholipids is regulated. Synthesis increases when

growth rate increases, is inhibited when starvation

occurs, and the fatty acid composition of the

membrane changes with growth temperature. Several

genes encoding enzymes involved in membrane

synthesis are located in the rpmF-plsX-fab operon. In

this operon, a gene encoding a phospholipid synthetic

gene of unknown function, plsX, lies just downstream

of the ribosomal protein gene rpmF and upstream of

five fatty acid biosynthetic genes, fabH, fabD, fabG,

acpP, and fabF. The operon is also complex;

transcription is initiated from at least eight promoters.

In addition, some transcripts produced by the operon

are cleaved by RNases while others terminate at one

of three specific points at the 5' end of plsX. This

work demonstrates that a weak transcriptional

terminator (an attenuator) lies at the 5' end of plsX.

The attenuator was localized to a 200 bp segment.

Analysis of the secondary structure of the attenuator

mRNA has lead to a model which includes four

stem-loop structures. In this model, the plsX start

codon lies within the loop of the second stem. Two

tandem stems are located directly upstream of the

mapped 3' endpoints. Mutational analysis shows that

all four stem-loops play a role in attenuator activity.

Regulation of the attenuator and the attenuator's

mechanism of controlling downstream gene expression

were investigated. Ribosome binding to attenuator

mRNA, the PlsX protein, ppGpp concentration, and

rate of lipid synthesis all appear to have no effect on

attenuator activity. Interestingly, growth temperature

appears to have an effect on both attenuator activity

and the activity of one or more of the promoters

upstream of rpmF, P1, P2, and P3. Activity of the

three promoters is 4.5-fold higher at 28*C as

compared with 42*C. The attenuator also appears to

increase expression of downstream genes 2-fold as

temperature decreases. Though the attenuator region

terminates transcription, growth

temperature-regulation of attenuator activity is

apparently mediated by a change in stability of the

mRNA. These data demonstrate that transcriptional

expression of plsX is 9-fold higher at 28*C as

compared with 42*C. The striking dependence on

temperature of plsX expression suggests a role for

PlsX in the temperature modulation of fatty acid

incorporation into the membrane phospholipids.

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