Chapter Questions
Diagram the elution pattern of the eukaryotic nuclear RNA polymerases from DEAE-Sephadex chromatography. Show what you would expect if you assayed the same fractions in the presence of $1 \mu \mathrm{g} / \mathrm{mL}$ of $\alpha$-amanitin.
Transcription of a class II gene starts at a guanosine $25 \mathrm{bp}$ downstream of the last base of the TATA box. You delete 20 bp of DNA between this guanosine and the TATA box and transfect cells with this mutated DNA. Will transcription still start at the same guanosine? If not, where? How would you locate the transcription start site?
Describe and give the results of an experiment that shows that polymerase I is located primarily in the nucleolus of the cell.
You suspect that a repeated sequence just upstream of a gene is acting as an enhancer. Describe and predict the results of an experiment you would run to test your hypothesis. Be sure your experiment shows that the sequence acts as an enhancer and not as a promoter element.
Describe and give the results of an experiment that shows that polymerase III makes tRNA and $5 \mathrm{~S}$ rRNA.
You are investigating a new class II promoter, but you can find no familiar sequences. Design an experiment to locate the promoter sequences, and show sample results.
Describe a primer extension assay you could use to define the $3^{\prime}$-end of the $5 S$ rRNA promoter.
How many subunits does yeast RNA polymerase II have? Which of these are "core" subunits? How many subunits are common to all three nuclear RNA polymerases?
Describe how epitope tagging can be used to purify polymerase II from yeast in one step.
Some preparations of polymerase II show three different forms of the largest subunit (RPB1). Give the names of these subunits and show their relative positions after SDSPAGE. What are the differences among these subunits? Present evidence for these conclusions.
What is the structure of the CTD of RPB1?
Draw a rough diagram of the structure of yeast RNA polymerase II. Show where the DNA lies, and provide another piece of evidence that supports this location for DNA. Also, show the location of the active site.
How many $\mathrm{Mg}^{2+}$ ions are proposed to participate in catalysis at the active center of RNA polymerases? Why is one of these metal ions difficult to see in the crystal structure of yeast RNA polymerase II?
Cite evidence to support pore 1 as the likely exit point for RNA extrusion during polymerase II backtracking.
What is meant by the term "processive transcription?" What part of the polymerase II structure ensures processivity?
What is the probable function of the rudder of polymerase II?
What is the probable function of the bridge helix? What is the relationship of $\alpha$-amanitin to this function?
What are the E site and A site of RNA polymerase II? What roles are they thought to play in nucleotide selection?
What role does the polymerase II trigger loop play in nucleotide selection? Illustrate with a schematic diagram of contacts to the base, sugar, and triphosphate.
What role does the Rpb4/7 complex play in opening or closing the clamp of RNA polymerase II? What evidence supports this role?
The 12 -subunit RNA polymerase II interacts with promoter DNA. What implications does this have for the state of the promoter DNA with which the polymerase must interact?
Draw a diagram of a composite polymerase II promoter, showing all of the types of elements it could have.
What kinds of genes tend to have TATA boxes? What kinds of genes tend not to have them?
What is the probable relationship between TATA boxes and DPEs?
What are the two most likely effects of removing the TATA box from a class II promoter?
Describe the process of linker scanning. What kind of information does it give?
List two common proximal promoter elements of class II promoters. How do they differ from core promoter elements?
Diagram a typical class I promoter.
How were the elements of class I promoters discovered? Present experimental results.
Describe and give the results of an experiment that shows the importance of spacing between the elements of a class I promoter.
Compare and contrast (with diagrams) the classical and nonclassical class III promoters. Give an example of each.
Diagram the structures of the U1 and U6 snRNA promoters. Which RNA polymerase transcribes each? What is the effect of moving the TATA box from one of these promoters to the other? Why does this seem paradoxical?
Describe and give the results of an experiment that locates the 5 '-border of the $5 \mathrm{~S}$ rRNA gene's promoter.
Explain the fact that enhancer activity is tissue-specific.