The Role of the Escherichia coli RNA Polymerase ?´Jaw in Transcription

Sammanfattning: 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 ( ?´), 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 ?´ 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.