Deck 9: Eukaryotic RNA Polymerasesand Their Promoters

Question 1

Transcription of a class II gene starts at a guanosine 25 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

Answer 1

No, transcription will not start in the same place.  It will start about 20 bp downstream of its normal start site.  You can locate the new start site with either S1 mapping or primer extension analysis.  The latter method will give better resolution

Question 2

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 g/ml of -amanitin

Answer 2

It was believed previously that in eukaryotic nuclei at least two RNA polymerases operate. One of these functions in transcribing major ribosomal genes and one or more function in transcribing the nuclear genes. Roeder and Rutter, in 1969 found out that there are three RNA polymerases.
The three RNA polymerases have distinct roles in transcription and each of the enzyme makes different kinds of RNA. These three enzymes were separated by the scientists using DEAE-Sephadex ion-exchange chromatography. The separation on the chromatographic column was possible as the three polymerases exhibit different properties and behaviors. The responses of these enzymes differ for the ionic strength and divalent metals in the column.
The separation was carried out by subjecting extracts from sea urchin embryos to DEAE-Sephadex ion-exchange chromatography. The diagram/separation graph from three RNA polymerases is as given below.
Diagrammatic representation of the separation of three RNA polymerases by ion-exchange chromatography - Thus, three RNA polymerases differ in their roles, properties and behaviors.

Question 3

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.

Answer 3

First the assumed enhancer arouses transcription. To perform that, first clone the gene, together with the enhancer and enhancer can be removed via cutting on either side of it having the constraint enzyme plus ligating DNA plasmid back together. Subsequently, reporter gene is connected to the promoter, therefore, you might examine for the efficiency of transcription.
At that point keep plasmid in the cells which usually express the gene, subsequently permitting time for translation as well as transcription, debris should be spun down plus test the supernatant activity of $-galactosidase. If the element of DNA certainly performs like enhancer, activity of $-galactosidase must reduce intensely once the removal of elements took place. Then this performance is also well-suited having the acting of element as the promoter element.
To rule that prospect out, do two tests:
• Initially remove the element and place it back in the plasmid in the opposite direction. If transcription keeps high, the element is independent form direction, that is the symbol of an enhancer but not the promoter.
• Secondly, eradicate the element keep it hundreds of base-pairs away from the start of transcription, either downstream or upstream of promoter. If transcription keeps high, the element is independent form direction, that is an enhancer's symbol but not the promoter.
Lastly, invert and move the element just to double check its position and direction independence.

Question 4

Describe and give the results of an experiment that shows that polymerase I is located primarily in the nucleolus of the cell.

Question 5

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.

Question 6

Describe and give the results of an experiment that shows that polymerase III makes tRNA and 5s rRNA

Question 7

Describe a primer extension assay you could use to define the 3-end of the 5S rRNA promoter.

Question 8

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

Question 9

Describe how epitope tagging can be used to purify Polymerase II from yeast in one step

Question 10

Some preparations of Polymerase II show three different forms of the largest subunit. Give the names of these subunits and show their relative positions after SDS-PAGE. What are the differences among these subunits Present evidence for these conclusions

Question 11

What is the structure of the CTD of RPB1

Question 12

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 this active site.

Question 13

How many Mg 2+ ions are proposed to participate in catalysis at the active center of RNA polymerase Why is one of these metal ions difficult to see in the crystal structure of yeast RNA polymerase II

Question 14

Cite evidence to support pore 1 as the likely exit point for RNA extrusion during Polymerase II backtracking

Question 15

What is meant by the term "processive transcription" What part of the Polymerase II structure ensures processivity

Question 16

What is the probable function of the rudder of Polymerase II

Question 17

What is the probable function of the bridge helix What is the relationship of -amanitin to this function

Question 18

What are the E site and A site of RNA Polymerase II What roles are they thought to play in nucleotide selection

Question 19

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

Question 20

What role does the Rpb4/7 complex play in opening or closing the clamp of RNA Polymerase II What evidence supports this role

Question 21

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

Question 22

Draw a diagram of a Polymerase II promoter, showing all of the types of elements it could have.

Question 23

What kinds of genes tend to have TATA boxes What kinds of genes tend not to have them

Question 24

What is the probable relationship between TATA boxes and DPEs

Question 25

What are the two most likely effects of removing the TATA box from a class II promoter

Question 26

Diagram the process of linker scanning. What kind of information does it give

Question 27

List two common upstream elements of class II promoters. How do they differ from core promoter elements

Question 28

Diagram a typical class I promoter.

Question 29

How were the elements of class I promoters discovered Present experimental results.

Question 30

Describe and give the results of an experiment that shows the importance of spacing between the elements of a class I promoter.

Question 31

Compare and contrast (with diagrams) the classical and nonclassical class III promoters. Give an example of each

Question 32

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

Question 33

Describe and give the results of experiments that locate the 5' border of the 5S rRNA gene's promoter.

Question 34

Explain the fact that enhancer activity is tissue specific.