Antitermination is operative in bacteriophage T7 and is largely dependent on one promoter

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Antitermination is operative in bacteriophage T7 and is largely dependent on one promoter

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Title: Antitermination is operative in bacteriophage T7 and is largely dependent on one promoter
Author: Robins, William Paul
Abstract: The translocation of the T7 genome into the cell is a multistep process. Following adsorption, approximately 850bp of the 40kb linear genome is internalized to expose host-specific promoters in the leading end to transcription components. There are three strong early promoters, PA1, PA2, and PA3 on this leading 850 bp. Further T7 genome internalization is coupled to transcription and I have measured internalization rates to characterize the rate of transcription by E. coli RNA polymerase in vivo. E.coli RNAP internalizes the entire 40kb and distal parts of the genome are internalized nearly as efficiently and at the same rate as the leading end. I have shown that processivity is dependent on the antitermination element boxA, located 63 bp downstream from PA3, and on only one of the three early promoters. However, when any one of boxA, PA3, or the host antitermination factor nusB is mutated the efficiency, rate, and apparent processivity of transcription -- and thus the efficiency of genome internalization are all significantly reduced. The PA3 promoter, boxA, and E. coli nusB are all non-essential for T7 growth, but they confer a fitness benefit to wild-type phage by increasing the rate of genome internalization. In T7, the minimal requirement for antitermination is promoter PA3 and the boxA sequence. I have found that transcripts initiating at PA1 and PA2 are not effectively antiterminated by boxA, however those from PA3 alone do. Upon further investigation it was shown that there is a requirement for sequences upstream of the -35 hexamer of PA3 to confer full antitermination. After T7 expresses its own single-subunit RNA polymerase, bacteriophage T7 must shutoff host transcription via the phage proteins gp0.7 and gp2. In the absence of host RNAP shutoff, T7 DNA is degraded and the infection fails. I have found that the absence of either promoter PA3 or boxA,gene 2 is unnecessary for growth. These results argue the target for shutoff is actually antiterminating transcription.
Department: Cellular and Molecular Biology
Subject: Bacteriophages Escherichia coli--Genetics Genetic transcription
URI: http://hdl.handle.net/2152/18110
Date: 2008-08

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