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Title: Studies on the mechanism of termination of transcription
Author: O'Hare, Kevin M.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1978
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The termination of RNA synthesis by purified E.coli, RNA polymerase, at the principal termination site for host-mediated early transcription of coliphage T7 DNA, has been studied in detail. Recognition of the "stop" signal and cessation of RNA chain growth are highly efficient. A nitrocellulose binding assay has shown that the subsequent release of RNA product is rapid, but unexpectedly the separation of DNA and enzyme is slow, such that it becomes rate limiting for further transcription. Conditions which inhibit DNA release have been defined so that enzyme remains bound at or near the termination site. Digestion of the DNA by restriction endonucleases followed by filtration on nitrocellulose has then allowed the identification and enrichment of restriction fragments which contain the termination site. This affords a novel route for mapping restriction sites near this and perhaps other termination sites. Using an antiserum prepared against purified sigma protein, the possible involvement of sigma in termination has been investigated. It was hypothesised that sigma might bind to core enzyme at or near the termination site, and might have an essential role in some or all steps in termination. The results indicate that sigma has no such role in the termination of transcription in vitro. Preliminary studies with the transcriptional termination factor rho suggest that rho may increase the rate of RNA release, but does not affect the rate of DNA release. Attempts were made to extend the known nucleotide sequence around this termination site. RNA was synthesised in vitro, hybridised to the complementary single strand of DNA and used as a primer for DNA polymerase I of E.coli. However, DNA synthesis at nicks and ends of DNA so obscured the results that only tentative progress was made in determining the sequence of the DNA formed by extension of the RNA primer, and in locating restriction sites within this DNA.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available