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- Ederth, Josefine, 1974-
(author)
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The Role of the Escherichia coli RNA Polymerase β´Jaw in Transcription
- 2004
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Doctoral thesis (other academic/artistic)abstract
- Transcription, the central step in gene expression and regulation, is carried out by the DNA-dependent RNA polymerase (RNAP). Bacterial RNAP is a complex molecular machine in which the network of interacting parts and their movements, including contacts to nascent RNA and the DNA template, are at best partially understood.In this work, the aim has been to elucidate the role of the RNAP jaw-domain, which is part of the RNAPs largest subunit ( β´), in transcription. The jaw-domain is interesting because of its close proximity to downstream DNA in the complex. Downstream DNA is known as a component involved in all three steps of transcription. However, the key role it plays in regulating the enzyme´s activity is far from understood.Initially, two novel mutations in the β´ jaw-domain were isolated and characterized in vivo. It was shown that the jaw-domain has specific effects on regulation of the ColE1 plasmid copy number, likely via different effects on initiation at the RNA Iand RNA II- promoters.Further, in vitro characterisations indicate that contacts of the jaw-domain to downstream DNA, at the leading edge of the transcription complex, contribute to regulation during all three phases of transcription. The results provide insight into the role of the jaw-domain-downstream DNA contact in transcription initiation and pausing, and suggest possible explanations for the previously reported effects on ColEI plasmid copy number.The RNAP jaw-domain is situated at ~30 Å distance from the active site. Suppressor mutations were selected to gain further knowledge of how the jaw-domain can exert such profound effects on the enzyme´s activity. Allele specific suppressor mutations were found in the rifampicin-pocket, only a few Ångströms away from the active site. It is shown that the suppressor substitutions compensate for the jawdeletion RNAPs defects in transcriptional pausing and inability to grow at elevated temperatures. These same substitutions exacerbate the jaw-deletion´s defect at transcription initiation, which suggests that the elongation defects are responsible for the temperature sensitive growth phenotype of the jaw-deletion strain. In light of the RNAP crystal structures available, a possible mechanism for counteracting effects on the active site mediated by the suppressor mutations and the jaw-deletion is suggested.
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| 2. |
- Åberg, Anna, 1977-
(author)
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New insights into the role of ppGpp and DksA through their effect on transcriptional regulation of housekeeping and colonization related genes of Escherichia coli
- 2008
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Doctoral thesis (other academic/artistic)abstract
- Bacteria have the ability to sense different environmental signals. When an environmental stress is detected, bacteria rapidly adjust their gene expression profile to be able to survive and thrive. The transduction of such environmental signals often requires the coordinated involvement of several factors that constitute complex regulatory networks. Hence, depending on the combination of signals, a unique gene expression profile required to adapt to the specific stress conditions is generated. Proteins are the best-known regulatory factors. However, non-proteinaceous molecules are also important in signal-responsive control of bacterial gene expression. Alarmones are low molecular weight non-proteinaceous regulatory factors which can characteristically be rapidly turned-over to mediate instant changes in gene expression. One such alarmone is the modified nucleotide ppGpp, which directly binds to RNA polymerase to alter its activity. The levels of this alarmone are expected to rapidly increase in response to any environmental stress that result in slow proliferation. DksA, a putative ppGpp co-regulator that likewise directly targets RNA polymerase, has been suggested to be required for both the positive and negative regulation mediated by ppGpp in Escherichia coli. This thesis describes dissection of the role of ppGpp and DksA on transcriptional regulation, primarily using the fim genetic determinant that encodes for the type 1 fimbriae. Type 1 fimbriae are involved in adhesion to abiotic surface and initial adhesion/invasion of bladder cells, as well as in biofilm formation. We found that ppGpp regulates phase variation by increasing the sub-population of cells that express the fimbriae. The effect of ppGpp was ultimately traced to its role in transcription of the fimB gene that encodes a recombinase involved in the phase variation process (paper 1). In contrast, we unexpectedly found that lack of DksA causes an increase, rather than a decrease, in transcription from the fimB P2 promoter in vivo. However, in vitro transcription studies demonstrated that ppGpp and DksA, both independently and co-dependently, stimulate transcription from the fimB P2 promoter. These seemingly contradictory results from the in vivo and in vitro transcriptional studies were shown to be, at least in part, a consequence of the increased association of Gre-factors with RNA polymerase that can occur in the absence of DksA in vivo (paper 2). The results outlined above have implications for the role of ppGpp and/or DksA in global gene expression. Using gene expression profile (microarray analysis) during the transition from logarithmic to stationary phase of E. coli, we found that while most of the genes regulated by ppGpp and DksA are regulated in the same direction by the two factors, many were not. In addition to the fim genes, genes involved in flagella functioning, taxis responses, and a few genes encoding different transport systems are also differentially regulated in ppGpp- and DksA-deficient strains in vivo. Our results clearly indicate that the effect of deficiencies in ppGpp and DksA is far more complex than phenotypic similarity of the corresponding mutants anticipated by the proposed concerted action of ppGpp and DksA on gene expression (paper 2 & 3).
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