Gen511-Homework4-21-08-key

Gen511-Homework4-21-08-key - 1 Gen 511: Homework Assignment...

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Gen 511: Homework Assignment Due 4/21/2008: “When Polymerases Collide” Homework assignment is based on the following paper: Liu et al., 1993. The DNA replication fork can pass RNA polymerase without displacing the nascent transcript. Nature 366, 33-39. A PDF of the paper is posted on WebCT. Answer the questions in italics regarding Figure 1 and Figure 2. Figure 1. Critique this experiment. Do you see any potential flaw in this experiment or the conclusions? How could the experiment be improved? The sketch accompanying the figure shows three possible scenarios for what could happen when the DNA polymerase collides with the stalled RNA polymerase: displace, stall, or pass. Displace and pass both generate long replicated DNA molecules, produced by extension of the 3283 bp plasmid; these are easily visualized as the smears of lengthening DNA on the gel. However, stall would produce a much shorter DNA molecule, because replication begins at a nick only 170 nucleotides upstream of the stalled RNA polymerase. There is no evidence for any “stalled” DNA polymerase; i.e. no signal on the gel near the arrow marked “unit length plasmid”. If some proportion of the DNA polymerase molecules did stall at the site of the stalled RNA polymerase, would we be able to see it in the gel? Perhaps not. The molecules we see on the gel are labeled with radioactive dTTP. Since DNA replication begins only 170 nucleotides upstream of the stalled RNA polymerase, then the only way the “stalled” DNA could be labeled is by the incorporation of dTTP into the 170 nucleotide segment. We don’t know if enough labeled T’s can be incorporated into that segment in order to produce a signal visible on the gel. In contrast, the long DNA produced by bypassing the RNA polymerase has thousands of
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Gen511-Homework4-21-08-key - 1 Gen 511: Homework Assignment...

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