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Unformatted text preview: Answers to Weaver end of chapter questions Chapter 6 The Mechanism of Transcription in Prokaryotes 1. The E. coli core RNA polymerase is capable of binding only loosely and non-specifically to promoters. It will bind T4 phage DNA non-specifically and will transcribe it only weakly. Addition of the σ subunit will add specificity and lead to tight binding of RNA polymerase to T4 phage DNA, which is recognized by E. coli RNA polymerase. Interestingly, core RNA polymerase transcribes highly nicked DNA templates very well but this is an artifact. This explains the efficient transcription of a nicked calf thymus DNA but this is an artifact and is non-specific. Since E. coli RNA polymerase does not recognize eukaryotic calf thymus DNA, addition of the σ subunit will not result in tight binding of the polymerase and the transcriptional efficiency will not change. 2. Bautz et al. used hybridization competition to demonstrate that the E. coli holoenzyme is specific for the phage T4 immediate early genes whereas the core is non-specific. In this experiment, phage DNA was immobilized on a filter and hybridized with labeled RNA transcribed using either the core enzyme or the holoenzyme. Whether the labeled RNA transcribed by each enzyme was from immediate early genes, or from all phage genes, was determined by testing the ability of unlabeled competitor RNA to compete for binding to the labeled DNA. Binding of the RNA transcribed by the holoenzyme was competed to a large extent by RNA transcribed from immediate early genes and to a lesser extent by other phage RNA. The RNA transcribed by the core enzyme was competed to about the same extent with immediate early, delayed early and late T4 RNA’s suggesting that the RNA transcribed by the core enzyme represented all three classes of genes. (Note: The material needed to answer this question has been deleted from the 4 th edition, so students will need extra preparation if this question is assigned.) 3. Using phage T4, Bautz et al. determined that E. coli core RNA polymerase is indiscriminate about which DNA strand it transcribes. The first step was isolation of phage RNA generated in vivo. Since authentic phage RNA is made asymmetrically, only one strand in any given region was represented in this sample. This RNA was hybridized with labeled phage RNA transcribed in vitro using either the core polymerase or the holoenzyme. Single-stranded unhybridized RNA was digested with RNase and the amount of radiolabeled hybrids was quantified. They predicted that, if a particular enzyme transcribes RNA in vitro in a similar fashion to its transcription in vivo, the strands would all be the same sense and would not hybridize. If on the other hand an enzyme transcribes T4 phage DNA indiscriminately in vitro, some sequences would be antisense to the authentic phage RNA, and double stranded, RNase-resistant hybrids would be formed. They observed double stranded RNase resistant hybrids only when the core enzyme was used to transcribe the in...
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