Amino acid deprivation of Escherichia coli results in the accumulation of
guanosine 5'-triphosphate 3'-diphosphate and guanosine 3', 5'-bispyrophosphate,
collectively designated (p)ppGpp. These nucleotides are synthesized by a
ribosome-associated enzyme encoded by the relA gene and are thought to
represent starvation stress signal molecules. They may mediate the global
reorganization of cellular metabolism, known as the stringent response, that is
characteristic of starving bacteria and which apparently represents a survival
strategy. In this dissertation, the following aspects of the stringent response are
characterized: (i) the temperature phenotypes of relA mutants; (ii) the C-terminal
domain of RelA; and (iii) the role of RelC (ribosomal protein L11) in the regulation
of RelA.
All three of the commonly used relA mutant alleles of E. coli, relA1, relA2,
and ∆relA251::kan, conferred temperature-sensitive (ts) phenotypes. The
temperature sensitivity was associated with decreased thermotolerance, and relA
mutants were killed at temperatures as low as 42°C. The ts phenotypes were
suppressed by increasing the osmolarity of growth media and by certain mutant
alleles of rpoB, the gene encoding the β-subunit of RNA polymerase, suggesting
a defect in transcription. DNA in heat-shocked wild type bacteria was initially
relaxed but the normal level of negative supercoiling was restored within 10 min
after heat shock. In contrast, DNA in heat-shocked relA mutants remained
relaxed. This relA-associated defect in DNA negative supercoiling was
suppressed by increased medium osmolarity. Furthermore, the re/A-mediated ts
phenotype was suppressed by low concentrations of novobiocin, a specific
inhibitor of the B subunit of DNA gyrase. Moreover, low concentrations of
novobiocin restored DNA negative supercoiling in the relA mutant at high
temperature. Based on previous reports, it is proposed that low concentrations of
novobiocin induce the synthesis of the DNA gyrase A and B subunits, and the
resulting increase in DNA gyrase activity restores normal supercoiling at high
temperature. Collectively, the data suggest that relA mutants are unable to
efficiently transcribe key genes required for thermotolerance, and this defect is
related to their inability to restore negative supercoiling of DNA at higher
temperatures. In addition, the proposed defect in transcription may be related to
the observation that ppGpp binds to the p-subunit of RNA polymerase.
The portion of relA encoding the C-terminal half of RelA (starting at amino
acid 455), designated 'RelA, was subcloned. Overexpression of 'RelA relaxed the
stringent response by inhibiting (p)ppGpp synthesis during amino acid
deprivation. 'RelA represented the ribosome-binding domain, and when
overexpressed, 'RelA somehow replaced RelA on ribosomes. The 'RelA
ribosome-binding domain was further localized to a region between amino acids
455 to 682 with the main binding activity in a fragment extending from amino
acids 560 to 682. Several criteria were used to establish the fact that 'RelA also
mediated the formation of homodimers. These included co-purification of RelA
and 'RelA, glutaraldehyde protein crosslinking, and analysis by nondenaturing
polyacrylamide gel electrophoresis. The dimerization domain overlapped with
the ribosome-binding domain. Affinity blotting assays using 'RelA as a probe
revealed RelA and 'RelA as the only proteins in crude cell extracts that bound
'RelA. Therefore, these studies failed to identify the ribosomal components that
interact with RelA.
Amino add-deprived rplK (previously known as relC) mutants of E. coli
cannot activate ribosome-bound RelA and consequently exhibit relaxed
phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11
is involved in regulating the activity of RelA. The overexpression of derivatives of
rplK that contained short N-terminal deletions that eliminated the proline-rich
helix resulted in relaxed phenotypes. In contrast, bacteria overexpressing normal
L11 exhibited a typical stringent response. The L11 mutant proteins were
incorporated into ribosomes. A derivative in which Pro22 was changed to Leu22
was constructed by site-directed mutagenesis. This amino add substitution was
sufficient to confer a relaxed phenotype when it was overexpressed. A variety of
methods were used in attempts to demonstrate a direct interaction between L11
and RelA, but all yielded negative results. These results indicate that the N-terminal
proline-rich helix, and Pro22 in particular, is directly involved in activating
RelA activity during amino acid deprivation. The mechanism apparently does not
involve a direct interaction between RelA and L11 and is presumably mediated
by another ribosomal component. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/9916 |
| Date | 16 August 2018 |
| Creators | Yang, Xiaoming |
| Contributors | Ishiguro, Edward E. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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