| 1. |
- Croitoru, Victor, 1976-
(författare)
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Study on the Function of Translation Initiation Factor IF1
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Initiation is the first step in protein biosynthesis representing a fundamental event in cell life which determines fidelity, efficiency and regulation of gene expression. In addition to the ribosome and mRNA, three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. Several minor functions have been attributed to the smallest of these factors, IF1. However, the main function of IF1 remains to be elucidated.In order to investigate the role of this protein in the initiation process we have mutated the corresponding gene infA. Using a high-copy plasmid and site-directed mutagenesis, the six arginine residues of IF1 were separately altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. This strategy was followed by deletion of the chromosomal infA gene. All variants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Several of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type allele. Some of these mutants displayed reduced growth rates and a partial cold-sensitive phenotype.The influence of the leucine group of mutants in IF1 on the expression of two reporter genes with different initiation and/or +2 codons has been investigated. Our results do not indicate any involvement of IF1 in recognition of the +2 codon immediately following the start codon, thus representing the A-site. In addition, this group of mutants has no changed efficiency of decoding at the near-cognate initiation codons UUG and GUG. However, one cold-sensitive IF1 mutant shows a general overexpression of both reporter genes, in particular at low temperatures. Overall, the results do not support the hypothesis that IF1 could possess codon discriminatory functions while blocking the A-site of the ribosome.In this study we also identify that IF1 has RNA chaperone activity both in vitro and in vivo. The chaperone assays are based on splicing of the group I intron in the thymidylate synthase gene (td) from phage T4. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.
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| 2. |
- Ederth, Josefine, 1974-
(författare)
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The Role of the Escherichia coli RNA Polymerase β´Jaw in Transcription
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 3. |
- Vega Riquelme, Carmen Paulina, 1983-
(författare)
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Nitrate stable isotopes and major ions in snow and ice from Svalbard
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Increasing atmospheric reactive nitrogen (Nr), as consequence of human activities, has generated accumulation of nitrate (NO3-) in Arctic regions. The Arctic has fragile nitrogen limited ecosystems that can be altered by increases of dry or wet deposition of Nr. Ice cores have shown increments of twofold in nitrogen deposition over Greenland and Svalbard during the 20th century. Ice core NO3- stable isotopes, (δ15N-NO3- and δ18O-NO3-), have the potential to serve as proxy of nitrogen oxides (NOx) sources and atmospheric oxidation pathways. NO3- is difficult to interpret in ice since it has several sources and experiences post-depositional processes, e.g. photolysis, relocation, evaporation and diffusion. The present work shows the results of NO3- and NO3- stable isotopes analyses of ice cores, snow and precipitation from Svalbard, in order to obtain records of natural and anthropogenic sources of NO3-. In addition, meltwater percolation effects on the snowpack ion content were also studied. A comparison between NO3- records from different Svalbard ice cores and NOx and SOx emission profiles from different regions shows that the major source regions affecting Svalbard are Western Europe and North America, followed by Central Europe and former USSR. Post-1950s δ15N-NO3- measured at Lomonosovfonna is influenced mainly by fossil fuel combustion, soil emissions, and forest fires. There is an east-west gradient in snow NO3- stable isotopes during 2010/2011, with lower δ15N-NO3- and higher δ18O-NO3- values at eastern sites; the results evidence differences in the origin of air masses arriving at Svalbard sites, mainly Eurasia, to the eastern sites, and Northern Europe to the western sites. The effects of post-depositional change on the ice core chemistry were studied, finding that 45% of annual snowpack suffers melt at Lomonosovfonna during the last 60 years. Percolation lengths were estimated as ≈1 m for most of ions; therefore, it is expected that the atmospheric ionic signal is preserved at annual or bi-annual resolution within the last 60 years at the Lomonosovfonna summit. The results presented here suggest that NO3- stable isotopes from Svalbard ice cores are useful to describe different sources and source regions of NOx, contributing to the assessment of nitrogen enrichment for this region.
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