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A. There is a GTPase activating protein (GAP) that interacts with my protein and activates its GTPase activity. This GAP is in turn activated by exposure of cells to adrenaline, so the GTPase activity of my protein is highly stimulated by adrenaline. The reason I only see GDP-bound protein under these circumstances is that as soon as my protein binds to GTP it hyrolyzes it to GDP. In the absence of adrenaline the GAP protein is inactive, and the GTPase activity of my protein is very low. I can therefore isolate my protein bound to GTP.

B. There is a nucleotide exchange factor (NEF) that interacts with my protein and catalyzes its exchange of GDP for GTP. This NEF is in turn repressed by exposure of cells to adrenaline. Under these conditions, then, my protein can hydrolyze GTP to GDP, but this GDP then remains bound to my protein and is not exchanged for GTP. Thus when I isolate my protein I isolate it in its GDP-bound form. In the absence of adrenaline the NEF is active, so that when my protein hydrolyzes GTP to GDP, this GDP is rapidly exhanged for GTP. Thus when I isolate my protein I isolate it in its GTP-bound form.

2. If male (XY) Drosophila embryos carried a DNA construct encoding an inducible sex-lethal gene lacking the 3rd exon, and expression of this gene were induced early in development, what would you predict would be the phenotypic effect? What if the DNA construct encoded an inducible transformer gene lacking its second exon?

In the first case, the male embryos would express functional sex-lethal protein upon induction of the construct. The sex-lethal protein could then promote expression of functional transformer protein by blocking recognition of transformer's second exon, which carries a premature stop codon. Transformer could then stimulate production of a female-specific version of the double-sex protein, by stimulating recognition of double-sex's 4th exon. Double-sex would then repress transcription of male-specific genes, and female sexual development would occur.

In the second case, the male embryos would not express functional sex-lethal protein. However, they would express functional transformer protein, and transformer would in turn stimulate production of the female-specific version of the double-sex protein, which would repress transcription of male-specific genes. Again, female sexual development would occur.

3. Imagine that an in vitro transcription reaction could be used to synthesize RNA using UTP, CTP, GTP, and dATP (rather than ATP). If you exposed this in vitro-transcribed RNA to all of the components necessary for splicing, would splicing occur? Why or why not?

Splicing would not occur, because the 2' hydroxyl of the branch point adenosine is the "attacking" group in the first transesterification reaction catalyzed by the spliceosome. If this hydroxyl group is not present (as it is not in dATP), the reaction cannot occur.

[The RNA is also likely to have an aberrant structure, because the 2' hydroyxl groups along the chain are often involved in RNA folding.]

4. Predict the effects of mutating the DNA encoding a eukaryotic gene's poly-A signal so that it does not conform to the consensus recognition site for CPSF.

If CPSF does not recognize a new RNA's poly-A signal, it will not bind to the RNA and stimulate formation of the complex that catalyzes cleavage and poly-adenylation at that site. In turn, this is likely to block transcription termination at its usual location downstream from the poly-A site. Thus, transcription will continue through this region, presumably until another poly-A site is reached and transcribed.

5. Describe two different types of mutation to the DNA encoding a bacterial rho-independent terminator that would be likely to prevent transcriptional termination at that site.

A rho-independent terminator has two distinct structural elements that are thought to trigger termination. First, a tight stem-loop forms in the RNA and interacts with RNA polymerase, causing the polymerase to pause. Mutations that disrupted basepairing in the stem of this structure should block termination. Second, a stretch of U residues just downstream of the stem-loop region form weak basepairs with the DNA template, allowing the RNA to dissociate from the DNA while the polymerase is paused. Mutations which changed these U:A RNA/DNA basepairs to sequences with stronger interactions (e.g. G:C basepairs) should also block termination.

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