Deck 10: General Transcription Factorsin Eukaryotes

Question 1

You are studying a new class of eukaryotic promoters (class IV) recognized by a novel RNA polymerase IV. You discover two general transcription factors that are required for transcription from these promoters. Describe experiments you would perform to determine which, if any, is an assembly factor, and which is required to recruit the RNA polymerase to the promoter. Provide sample results of your experiments.

Answer 1

The hypothetical study containing class IV promoter recognized by RNA polymerase IV with the help of two general transcription factors require a special assembly formation. The experimental procedure necessary for determining this assembly has been described below.
It is important for the primary assembly factor to bind to the promoter independently and in turn facilitate the binding of other transcription factors. This query could be easily solved using a gel mobility shift experiment. The experiment must use both the transcription factors independently and in combination.
An ideal result would indicate the binding of a short labelled piece of DNA containing the promoter in presence of 1 st factor. The lane containing 2 nd factor will not have any shift since this factor would not bind independently. The lane containing both the factors will indicate a supershift, since the 1 st factor will facilitate the binding of a 2 nd factor.
This situation could also be explained using a DNase footprinting experiment. A footprint would be observed in the promoter region of the DNA due to the 1 st factor, which will be absent in case of a 2 nd factor. The addition of both factors, on the other hand, will cause the formation of a larger footprint than in case of 1 st factor alone.
The question regarding which factor is required for recruiting RNA polymerase IV, a gel mobility shift assay or DNase footprinting experiment (with a polymerase and both the factors) could be performed. In case the 2 nd factor is crucial for recruiting Pol IV, a supershift will be observed after the combination of 1 st and 2 nd factor, rather than due 1 st factor alone.
The results obtained will suggest that the 2 nd factor can recruit Pol IV only after the binding of a 1 st factor. This claim can also be strengthened using an in vitro transcription assay, where the transcription will only start in case of the mixture containing polymerase IV, and the combination of both the factors.

Question 2

List in order the proteins that assemble in vitro to form a class II preinitiation complex.

Answer 2

Transcription factors are the proteins required for assisting eukaryotic RNA polymerase in binding to the specific promoters. The class II preinitiation complex is formed by binding of RNA polymerase II with transcription factors. The sequence of binding of these factors is as given below.
The specific order of binding transcription factors is that initially TFIID with the help of TFIIA (optionally) joins with TATA box, thus forming a DA complex. This complex then binds with TFIIB. It is followed by the binding of TFIIF along with RNA polymerase at the position of -34 to +17. Finally, the factors TFIIE and TFIIH join to form DABPolIFEH preinitiation complex.

Question 3

You discover that one of your novel class IV transcription factors contains TBP. Describe an experiment you would perform to identify the TAFs in this factor.

Answer 3

The transcription factors may often contain a TATA box binding protein (TBP). It is important to identify the presence of TBP in this factor.
The identification of TBP in the factor may be identified using immuneprecipitation experiment. Let's assume that TBP is present in factor 1. This factor can be immunoprecipitated using an anti-TBP antibody. The precipitate obtained would then be treated using a denaturing agent such as 1M guanidine HCl. The TBP should then be re-precipitated with the antibody and subjected the supernatant with SDS PAGE. The proteins obtained in the supernatant would be the proteins associated with TBP in the transcription factor, and thus would be the TBP associated factors (TAFs).

Question 4

Describe and give the results of an experiment that shows that TFIID is the fundamental building block of the class II preinitiation complex

Question 5

Some of the class IV promoters contain two DNA elements (boxes X and Y), others contain just one (box X). Describe experiments you would perform to identify the TAFs that bind to each of these two types of promoters.

Question 6

Describe and give the results of an experiment that shows where TFIID binds together, but neither can bind independently to the preinitiation complex.

Question 7

You incubate cells with an inhibitor of the protein kinase activity of TFIIH and then perform in vitro transcription and DNase footprinting experiments. What step in transcription would you expect to see blocked What kind of assay would reveal such a blockage Would you still expect to see a footprint at the promoter Why or why not If so, how large would the footprint be, compared to the footprint in the absence of the inhibitor

Question 8

Describe and give the results of an experiment that shows that TFIID binds.

Question 9

You know that protein X and protein Y interact, but you want to know whether a particular domain of protein X interacts with protein Y, and if so, where. Design a hydroxyl radical cleavage analysis experiment to answer this question.

Question 10

Show the difference between footprints caused by the DAB and DABPolF complexes. What conclusion can you reach based on this difference

Question 11

Present a hypothesis that explains the fact that substitution of dCs for dTs and dIs for dAs, in the TATA box (making a CICI box) has no effect on TFIID binding. Provide the rationale for your hypothesis

Question 12

What shape does TBP have What is the geometry of interaction between TBP and the TATA box

Question 13

Describe and give the results of an experiment that shows TBP is required for transcription from all three classes of promoters.

Question 14

Describe and give the results of an experiment that shows that a class II promoter is more active in vitro with TFIID than with TBP

Question 15

Describe and give the results of an experiment that identifies the TAFs that bind to a class II promoter containing a TATA box, an initiator, and a downstream element

Question 16

Describe and give the results of a DNase footprinting experiment that shows how the footprint is expanded by TAF1 and TAF2 compared with TBP alone

Question 17

Draw a diagram of a model for the interaction of TBP (and other factors) with a TATA-less class II promoter

Question 18

Whole genome expression analysis indicates that yeast TAF1 is required for transcription of only 16% of yeast genes, and TAF9 is required for transcription of 67% of yeast genes. Provide a rationale for these results.

Question 19

Present examples of class II preinitiation complexes with:
a. An alternative TBP
b. A missing TAF
c. No TBP or TBP-like protein

Question 20

What are the apparent roles of TFIIA and TFIIB in transcription

Question 21

Draw a rough sketch of the TBP-TFIIB-RNA Polymerase II complex bound to DNA, showing only the relative positions of the proteins. How do these positions correlate with the apparent roles of the proteins Include an explanation of how TFIIB determines the direction of transcription.

Question 22

Describe and give the results of an experiment that mapped the sites on Rpb1 and Rpb2 that are in close contact with the finger and linker region of TFIIB

Question 23

Describe and give the results of an experiment that shows that TFIIH, but not the other general transcription factors, phosphorylates the IIA form of RNA polymerase II to the IIO form. In addition, include data that show that the other general transcription factors help TFIIH in this task

Question 24

Describe and give the results of an experiment that shows that TFIIH phosphorylates the CTD of polymerase II

Question 25

Describe an assay for DNA helicase and show how it can be used to demonstrate that TFIIH is associated with helicase activity.

Question 26

Describe a G-less cassette transcription assay and show how it can be used to demonstrate that the RAD25 DNA helicase activity associated with TFIIH is required for transcription in vitro.

Question 27

Draw a rough diagram of the class II preinitiation complex, showing the relative positions of the polymerase, the promoter DNA, TBP, TFIIB, E, F, and H. Show the direction of transcription.

Question 28

Describe and give the results of an experiment that shows that TFIIS stimulates transcription elongation by RNA polymerase II.

Question 29

Present a model for reversal of transcription arrest by TFIIS. What part of TFIIS participates most directly How

Question 30

Describe and give the results of an experiment that shows that TFIIS stimulates proofreading by RNA polymerase II

Question 31

What is the meaning of the term RNA polymerase II holoenzyme How does the holoenzyme differ from the core polymerase II

Question 32

Describe and give the results of an experiment that shows the effect of adding or removing a few base pairs between the core element and the transcription start site in a class I promoter.

Question 33

Which general transcription factor is the assembly factor in class I promoters In other words, which binds first and helps the other bind Describe a DNase footprinting experiment you would perform to prove this, and show idealized results, not necessarily those that Tjian and colleagues actually obtained. Make sure your diagrams indicate an effect of both transcription factors on the footprints

Question 34

Describe and give the results of copurification and immunoprecipitation experiments that show that SL1 contains TBP.

Question 35

Describe and give the results of an experiment that identified the TAFs in SL1.

Question 36

How do we know that TFIIIA is necessary for transcription of 5S rRNA but not tRNA genes

Question 37

Geiduschek and colleagues performed DNase footprinting with polymerase III plus TFIIIB and C and a tRNA gene. Show the results they obtained with: No added protein; polymerase and factors; and polymerase, factors and three of the four NTPs. What can you conclude from these results

Question 38

The classical class III genes have internal promoters. Nevertheless, TFIIIB and C together cause a footprint in a region upstream of the gene's coding region. Draw a diagram of the binding of these two factors that explains these observations.

Question 39

Draw a diagram of what happens to TFIIIB and C after polymerase III has begun transcribing a classical class III gene such as a tRNA gene. How does this explain how new polymerase III molecules can continue to transcribe the gene, even though factors may not remain bound to the internal promoter

Question 40

Describe and give the results of a DNAase footprint experiment that shows that TFIIIB + C, but not TFIIIC alone, can protect a region upstream of the transcription start site in a tRNA gene. Show also what happens to the footprint when you strip off TFIIIC with heparin

Question 41

Describe and give the results of an experiment that shows the following: Once TFIIIB binds to a classical class III gene, it can support multiple rounds of transcription, even after TFIIIC (or C and A) are stripped off the promoter

Question 42

Describe and give the results of an experiment that demonstrates the flexibility of TFIIIC in binding to boxes A and B that are close together or far apart in a class III promoter

Question 43

Diagram the preinitiation complexes with all three classes of TATA-less promoters. Identify the assembly factors in each case.