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to Answers Weaver end of chapter questions Chapter 11 General Transcription Factors in Eukaryotes 1. List in order the proteins that assemble in vitro to form a class II preinitiation complex. (a) TFIID with help from TFIIA (optional in vitro) binds to the TATA box forming the DA complex, (b) TFIIB binds next, (c) TFIIF along with RNA polymerase binds to region spanning from -34 to +17. (d) The remaining factors bind in order of TFIIE and TFIIH, forming the DABPolFEH preinitiation complex. 2. Describe and give the results of an experiment that shows that TFIID is the fundamental building block of the class II preinitiation complex. Refer to Fig. 11.1 Reinberg and colleagues performed gel mobility shift assays with different combinations of subunits of the preinitiation complex along with labeled DNA. Experiments without TFIID (lane 13, Fig 11.1a and lane 7, Fig 11.1b) resulted in no complex formation. 3. Describe and give the results of an experiment that shows where TFIID binds. Refer to Fig. 11.2. DNase I based footprinting shows that TFIID protects (binds) the small region just around the TATA box on both the template (Fig 11.2a, DNase I gel, lane labeled D) and non-template (Fig 11.2b, DNases I gel, lane labeled D) strands. 4. Describe and give the results of an experiment that shows that TFIIF and polymerase II bind together, but neither can bind independently to the preinitiation complex. Refer to Fig. 11.1. DNA gel mobility shift assays showed that when TFIIF was added to the DAB complex of transcription factors, there was no detectable difference between the resultant complex and the DAB complex; indicating that TFIIF was not able to bind the DNA (Fig 11.1 compare lanes 2 and 3). However, as increasing concentrations of Pol II was added along with TFIIF, new complexes appeared suggesting that TFIIF and RNA polymerase bind together to the DAB complex (Fig 11.1, lanes 3-7). Similarly, reactions containing decreasing amounts of TFIIF in the presence of Pol II resulted in increasing amounts of just the DAB complex (i.e. lowered higher order complex, Fig 11.1, lanes 8-12). 5. Show the difference between footprints caused by the DAB and DABPolF complexes. What conclusion can you reach based on this difference? Refer to Figs 11.2 and 11.3. The DAB complex forms on the TATA box protecting both the template and nontemplate strands of the DNA, although the DNA at position +10 appears to become hypersensitive to DNase I cleavage. Overall, DAB protects the TATA box region between -17 and -42, whereas DABPolF complex extends this protected region another 34 bases on the nontemplate strand, from position -17 to ~+17. The extended region of protection is consistent with the large size of Pol II and with its binding to one strand of the DNA. 6. 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. Refer to Fig. 11.5. TFIID binding to the TATA Box is dependent upon its TATA binding protein (TBP) subunit. (TFIID is composed of TBP and a number of TBP-associated factors (TAFs)). Since TBP binds in the minor groove of DNA, changes to the steric chemistry in the major groove should have no effect upon TFIID binding. The substitution of dC's and dIs alters the steric chemistry of the major groove but has no affect upon the minor groove and so the substitutions had no effect on TBP/TFIID binding. 7. What shape does TBP (TATA-box Binding Protein) have? What is the geometry of interaction between TBP and the TATA box? Refer to Fig. 11.6. The structure of TBP in the absence of DNA was saddle shaped with two "stirrups", suggesting that TBP sits on DNA (in the minor groove) like a saddle sits on a horse. The crystal structure solved in the presence of DNA template, however, revealed that the curved underside of the saddle instead of fitting neatly over the DNA, is roughly aligned with the long axis of the DNA. The curvature of the protein appears to force the DNA to bend almost 80 degrees, opening up the minor groove and almost straightening the helical twist in that region. 8. Describe and give the results of an experiment that shows TBP is required for transcription from all three classes of promoters. Refer to Fig. 11.7. Genetic experiments with temperature sensitive mutants of the TBP gene demonstrated that TBP is required for transcription from all three classes of promoters. In vitro transcription assays using Pol I, Pol II, and Pol III dependent promoters required an active TBP for transcription to occur. 9. 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. Refer to Fig. 11.10. Class II promoters contain a TATA box as well as Inr and DPE elements. When transcription from such a promoter is tested using an in vitro reconstituted transcription system containing either TBP or TFIID, the RNA products assayed by primer extension are greater (i.e. more intense bands by autoradiography) in reaction containing TFIID than TBP alone (see Fig 11.10, lanes 5 versus 6, and 7 versus 8).` 10. 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. Refer to Fig. 11.11. In order to determine which subunits (TAFs) of TFIID bind to the promoter, Tjian and colleagues photo-cross-linked TFIID with a radioactively labeled DNA fragment containing the Hsp70 promoter. Crosslinking covalently attaches the proteins that are intimately associated with (bound to) the DNA. Photocrosslinking is achieved by incorporating the nucleotide analogue, bromodeoxyuridine (BrdU) into the DNA and allowing TFIID to bind to the sequence. Irradiation of bound complexes with UV light cross-links protein(s) to the BrdU in the DNA. The DNA is then nuclease treated and the proteins are subjected to SDS-PAGE. Autoradiography of the gel derived from such an experiment showed that TAFII250 and TAFII150 became labeled, implying that these two proteins had been in close contact with the labeled DNA's major groove. 11. Describe and give the results of a DNase footprinting experiment that shows how the footprint is expanded by TAFII250 and 150 compared with TBP alone. Refer to Fig. 11.12. DNase footprinting showed that TBP caused a footprint only in the TATA box whereas the ternary complex caused an additional footprint in the initiator sequence. 12. Draw a diagram of a model for the interaction of TBP (and other factors) with a TATA-less class II promoter. Refer to Fig. 11.13 (b and c). TBP cannot bind by itself to a TATA-less promoter since it interacts directly with the TATA sequence. However, since it is part of TFIID and TATA-less promoters contain other sequence elements that the TAFs of TFIID can bind, TBP is carried along in any interactions with TATA-less promoters. For example, elements such as initiators and downstream promoter elements (DPEs) can bind TAF II250 and TAFII150 and secure TFIID (containing TBP) to the promoter. Alternatively, transcription factors that bind to specific sequence elements (i.e. GC boxes) and interacts with TFIID subunits would be able to recruit TFIID (with TBP) to TATA-less promoters. 13. Whole genome expression analysis indicates that yeast TAFII145 is required for transcription of only 16% of yeast genes, and TAFII17 is required for transcription of 67% of yeast genes. Provide a rationale for these results. TAFII17, is not only a subunit of TFIID, but is also a part of the transcription adapter complex known as SAGA (named for the three classes of proteins in contains: SPTs, AdAs, and GCN5, along with its enzymatic activity, histone acetyltransferase). SAGA also contains TBP, a number of TAFs, and appears to mediate the effects of certain transcription activator proteins. Thus, TAFII17 plays a role in several important transcription related complexes, and is therefore required for transcription of a larger set of genes than TAFII145 14. Present examples of class II preinitiation complexes with: a) An alternative TBP. TRF1 (TBP-related factor 1) in Drosophila melanogaster is an alternative TBP and stimulates transcription just like TBP. It binds to TFIIA and TFIIB, while having its own group of TRF-associated factors called nTAFs. TRF1 stimulates transcription of the Drosophila tudor gene which contains two distinct promoters: a downstream promoter with a TATA box and a second promoter about 77 bp upstream of the first and has a TC box recognized by a complex including TRF1. b) A missing TAFII. TAFII30 is found only in a fraction of human TFIIDs, and its presence correlates with its responsiveness to estrogen. c) No TBP or TBP-like protein. TBP-free TAFII-containing complex (TFTC) is able to sponsor preinitiation complex formation without any help from TFIID or TBP. TFTC and TFIID have strikingly similar 3-D structures. Both contain a groove large enough to accept a double-stranded DNA but TFTC has a projection at its top due to domain 5. TFIID lacks both the projection and the domain. 15. What are the apparent roles of TFIIA and TFIIB in transcription? Without TFIIA and TFIIB, the preinitiation complex cannot form and transcription cannot initiate. TFIIA apparently helps TFIID bind to the promoter. TFIIB serves as a linker between TFIID and TFIIF/polymerase II. It has two domains, one of which is responsible for binding to TFIID, the other for continuing the assembly of the preinitiation complex. It is essential for binding RNA polymerase because the polymerase-TFIIF complex will bind to the DAB complex, but not to the DA complex. TFIIB focuses the positioning of the transcription start site, first it achieves coarse positioning by binding to TBP and RNA polymerase at the TATA box. Then, upon, DNA unwinding, fine positioning of the start site is achieved by TFIIB-DNA interactions. 16. Draw a rough sketch of the TBP-TFIIB-Pol 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? Refer to Fig. 11.17. TFIIB contains two domains. TFIIBc (Carboxy-terminal domain) interacts with TBP and DNA at the TATA box, with the DNA bent by TBP appearing to wrap around TFIIBc and the polymerase. After the bend, the DNA extends straight towards TFIIB N (N-terminal domain), which lies near the active site of the polymerase. Thus, TFIIB, by bridging between TBP and RNA polymerase II, positions the polymerase active site to initiate the appropriate distance downstream of the TATA box. 17. Describe and give the results of an experiment that mapped the sites on RBP1 and RBP2 that are in close contact with the finger and linker region of TFIIB. Refer to Fig. 11.19. Interactions between domains of proteins can be examined using a combination of photo-cross linking and hydroxyl radical cleavage analysis. Hydroxyl radical cleavage analysis exploits cysteine residues that can be introduced into one of the (putatively) interacting proteins by site directed mutagenesis. To each cysteine, an iron-EDTA (Fe-BABE) is attached. This moiety can generate hydroxyl radicals that cleave protein chains within 15 angstroms of the cysteine. Thus, any portion of the interacting protein within 15 angstroms of the cysteine will be cleaved. The fragments generated after cleavage can be visualized by SDSPAGE and Western blotting. Using such a procedure, Chen and Hahn, determined the regions of Rpb1 and Rpb2 that are in close contact with the finger and linker regions of TFIIB. They determined that the finger and linker regions of TFIIB are close together in the preinitiation complex and that this part of TFIIB (i.e. TFIIB N) contacts RNA Pol II in the protrusion, wall, clamp, and fork regions of the polymerase, near the active site. 18. 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. Refer to Figs 11.20 and 11.21. Reinberg and colleagues incubated Pol IIA with various mixtures of transcription factors. They included radiolabeled ATP in the reactions to measure phosphorylation of the polymerase and analyzed their samples by gel mobility shift assays. First, they demonstrated that adding ATP had no effect on the mobility of the DAB, DABPolF, or DABPolFE complexes. However, with TFIIH and ATP, the resultant complex (DABPolFEH) displayed a gel shift consistent with phosphorylation of Pol IIA (Fig 11.20). Second, they demonstrated that TFIID, B, F, and E were insufficient to cause phosphorylation and that TFIIH alone is sufficient. However, phosphorylation of the polymerase by TFIIH is enhanced by the presence of the other transcription factors, particularly TFIIE. This was evident based upon a time course of radiolabeled ATP incorporation into Pol IIA (DBFHPol IIA complex) by TFIIH in the presence and absence of TFIIE (Fig 11.21 panels b and c). 19. Describe and give the results of an experiment that shows that TFIIH phosphorylates the CTD of polymerase II. Refer to Fig. 11.22. Reinberg and colleagues cleaved the CTD from the phosphorylated Pol IIa subunit using chymotrypsin (a protease). The products were separated by SDS-PAGE, and the fragment containing the radiolabeled phosphate was determined to be the CTD of Pol II. 20. Describe an assay for DNA helicase and show how it can be used to demonstrate that TFIIH is associated with helicase activity. Refer to Fig. 11.23. Helicase activity can be determined essentially by examining the unwinding of a radiolabeled strand from a double stranded substrate. The substrate consists of a small (single strand radiolabeled) piece of DNA that has been allowed to hybridize to its complementary region in a much larger unlabeled single stranded piece of DNA. Helicase acitivity, i.e. unwinding of the strands results in separation of the small and large single stranded pieces, which can be visualized by agarose gel electrophoresis of the products. Addition of the various general transcription factors (i.e. TFIIH), +/- ATP, to the substrate and electrophoretic analysis of the products can be used to demonstrate that TFIIH is associated with helicase activity. 21. 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. Refer to Fig. 11.24. The rad25-ts24 DNA helicase subunit mutation results in a temperature sensitive variant of TFIIH. The G-less cassette is a template containing a yeast TATA element upstream of a 400 bp region with no G's in the nontemplate strand. This template can be used to produce transcripts in the presence of ATP, CTP, and UTP (but no GTP) of 375 and 350 nt in length. Transcription assays performed at various temperatures using extracts from wild-type and the temperature sensitive mutant cells (rad25 versus rad25-ts24) demonstrated lower levels of transcription even at the permissive temperature and complete loss of product formation at elevated temperatures in the mutant as compared to the wild-type rad25 extract. 22. 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. Refer to Fig. 11.26 23. Describe and give the results of experiment an that shows that TFIIS stimulates transcription elongation by RNA polymerase II. Refer to Fig. 11.27. Reinberg and Roeder formed elongation complexes by incubating polymerase II with a DNA template and nucleotides, allowing initiation to occur. Heparin was then used to bind free polymerase and block new initiation from occurring. The addition of TFIIS enhanced the rate of RNA synthesis over buffer alone (control reaction) by 2-fold within ~3 min and by 2.6 fold after ~7 min. 24. Present a model for reversal of transcription arrest by TFIIS. What part of TFIIS participates most directly? How? Refer to Figs. 11.28 and 11.29. TFIIS activates the RNAse activity in RNA Pol II, which cleaves off the extruded part of the nascent RNA and creates a new 3'-terminus in the enzyme's active site. TFIIS consist of three domains, one of which features a zinc ribbon. This zinc ribbon and in particular two acidic residues at its tip participate directly in metal coordination at the active site of Pol II that would participate in ribonuclease activity. 25. Describe and give the results of an experiment that shows that TFIIS stimulates proofreading by RNA polymerase II. Refer to Fig. 11.30. Hawley and colleagues isolated unlabeled elongation complexes that were paused at a variety of sites close to the promoter. Next, the complexes were "walked" to a defined position in the presence of radioactive UTP to label the RNA in the complexes. ATP or GTP was then added to extend the RNA by one more base, to position +43. The base that should be incorporated at that position is an adenine, however in the absence of adenine but presence of guanine, the polymerase will incorporate it although at a reduced efficiency. Labeled transcripts were then (a) digested with RNase T1 to measure the misincorporation of G, or (b) chased into full length via the addition of all four nucleotides, then cleaved (with RNase T1) to measure the loss of G from position +43 by proofreading. Comparison of removal of misincorporated G (i.e. proofreading) in the absence and presence of TFIIS revealed stimulation of proofreading by TFIIS such that almost no misincorporated products could be detected. 26. What is the meaning of the term RNA polymerase II holoenzyme? holoenzyme differ from the core polymerase II? How does the The RNA polymerase II holoenzyme is a complex containing RNA polymerase II and a subset of general transcription factors along with other proteins that help assemble class II preinitiation complexes, it includes the core polymerase II. 27. 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. Refer to Fig. 11.31. Paul and colleagues made insertions and deletions of up to 5 bp between the TIF-IB binding site and the normal transcription start site of a rRNA promoter. Transcription still occurred; however, the initiation site moved upstream or downstream according to the number of base pairs added or deleted. Addition or subtraction of more than 5 bp, however, blocked transcriptional activity. 28. 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. Class I promoters are recognized by two transcription factors, a core binding factor and a UPE-binding factor (UBF). The core-binding factor is the fundamental transcription factor required to recruit RNA Pol I. UBF is the assembly factor, as it helps the core-binding factor bind to the core promoter element, although the degree of reliance on UBF varies considerably between organisms. For footprinting results refer to Fig. 11.32. 29. Describe and give the results of copurification and immunoprecipitation experiments that show that SL1 contains TBP. Refer to Fig, 11.34. Tjian and co-workers demonstrated that SL1 is composed of TBP and three TAFs. First, they purified human SL1 by several different procedures (heparin-agarose colum chromatography, glycerol gradient centrifugation, immunoprecipitation using TBP antibodies), using an S1 assay to locate the SL1 activity. The same fractions (i.e. purified SL1) were then assayed for the presence of TBP by Western blotting, confirming that TBP copurified and/or immunoprecipitated with SL1. 30. Describe and give the results of an experiment that identified the TAFs in SL1. Refer to Fig. 11.35 Human SL1 was immunoprecipitated with anti-TBP antibody and subjected to SDS-PAGE. The TAFs were released from the immunoprecipitate with guanidineHCl and subjected to SDS-PAGE. Three polypeptides, with molecular masses of 110, 63, and 48 kD were observed. Comparison with standards of known TAFs identified the SL1 components as being TAFI110, TAFI63, and TAFI48. 31. How do we know that TFIIIA is necessary for transcription of 5S rRNA but not tRNA genes? Refer to Fig. 11.36. Brown and colleagues performed transcription assays using extracts from various cells tested against a 5S rRNA promoter and a tRNA promoter in the absence and presence of an anti-TFIIIA antibody. The antibody blocked transcription from the 5S rRNA promoter but not from the tRNA promoter. Thus, TFIIIA appears to be necessary for 5S RNA but not tRNA promoter transcription. 32. 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? Refer to Fig. 11.37 (no added protein, lane c, polymerase + factors, lane a, polymerase + factors + four NTPs, lane b). These data indicated that the (crude) transcription factor preparation bound both the internal promoter and an upstream region (in the tRNA gene). Specifically, the factors and polymerase strongly protected box B of the internal promoter and the upstream region and weakly protected box A of the internal promoter. When the polymerase, factors, and three nucleotides were added, the polymerase shifted downstream and a new region overlapping box A was protected. However, the protection of the upstream region persisted even after the polymerase moved away. 33. 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. Refer to Fig. 11.39. TFIIIC recruits TFIIIB (along with TBP) upstream of the internal TFIIIC binding site. After RNA polymerase recruitment, and TFIIIB remains in place, even after the polymerase moves (to the right). Thus, DNase footprinting with TFIIIB (+ TFIIIC) results in a footprint in a region upstream of the gene's coding area (Fig. 11.38). 34. 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? Refer to Fig. 11.39. TFIIIC binds internal promoter promotes binding of TFIIIB - recruits RNA polymerase III. RNA Pol III begins transcription TFIIIB remains in place as polymerase moves right (transcribes), it may or may not remove TFIIIC from the internal promoter. As indicated, TFIIIB remains in place, and can recruit more Pol III for another round of transcription. 35. 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. Refer to Figs. 11.37 and 11.38. Geiduschek and colleagues performed footprinting experiments with a labeled tRNA gene and combinations of purified TFIIIB and C. TFIIC alone protected the internal promoter, especially box B but did not protect the upstream region. When both factors were present, both the upstream region and internal promoter were protected. TFIIIB by itself does not result in a footprint indicating that its binding is dependent on the presence of TFIIIC. However, upstream protection by TFIIIB survives heparin treatment, but the protection of boxes A and B (TFIIIC binding site) does not. Thus, even if TFIIIC is stripped off, the TFIIIB footprint remains. 36. 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. Refer to Fig. 11.40. Briefly, complexes formed by recruitment of TFIIIB by TFIIIC/A were stripped of TFIIIC/A by high salt or heparin. The stripped templates were purified away from the removed/unbound factors and tested for their ability to support transcription, which they could. 37. 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. Refer to Fig. 11.41. Sentenac and colleagues bound yeast TFIIIC to cloned tRNA genes having variable distances between their boxes A and B. The complexes were then visualized via scanning transmission electron microscopy. When the distance between boxes A and B was zero, TFIIIC appeared as a large blob on DNA. However, with increasing distance between boxes A and B (up to 74 bp apart, Fig. 11.41, panel d), TFIIIC appeared as two globular domains separated by a linker of increasing length between them. The combination of large size and stretchability allows TFIIIC to contact two widely separated promoter regions with its two globular domains. 38. Diagram the preinitiation complexes with all three classes of TATA-less promoters. Identify the assembly factors in each case. Refer to Fig. 11.42. Analytical Questions 1. An assembly factor should be able to bind independently to the promoter and facilitate the binding of the other factor. To probe this question, you could perform a gel mobility shift experiment with each of the general transcription factors independently, and both together. Let's imagine that factor #1 is an assembly factor. In that case, the gel mobility shift experiment should show a shift of a short, labeled piece of DNA containing the promoter in the presence of factor #1. Factor #2 should not bind by itself, so there should be no mobility shift in the presence of factor #2 alone. But factor #1 should allow factor #2 to bind, so there will be a supershift when both factors are added together. A DNase footprinting experiment could also be done to answer the same question. Factor #1 should make a footprint in the promoter DNA, but factor #2 by itself should not. However, Factors #1 and #2 should produce a larger footprint than factor #1 alone. Nitrocellulose filter binding experiments would also work, but they are not much used anymore, perhaps because of a greater chance of artifacts. To see which factor is required to recruit RNA polymerase IV to the promoter, you could perform a gel mobility shift or DNase footprinting experiment with the polymerase and each factor individually, or together. If factor #2 recruits the polymerase, then a supershift (or greatly enlarged footprint due to the polymerase) should not occur with factor #1 alone, but should occur with factors #1 and #2 together. Although factor #2 recruits the polymerase, it can't do that on its own because it needs the assembly factor (factor #1) in order to bind to the promoter. An in vitro transcription assay should also show no transcription until both factors plus polymerase IV have been added to the reaction. 2. Let's assume that factor #1 contains TBP. We could immunoprecipitate factor #1 with an anti-TBP antibody, then treat the precipitate with a denaturing agent like 1M guanidine-HCl, then re-precipitate TBP and the antibody and subject the supernatant to SDS-PAGE. The proteins in the supernatant should be the proteins that associated with TBP in the factor, which are TAFs, by definition. 3. You could used labeled DNAs containing BrdU incorporated into one or the other of the two boxes, in DNA photo-cross-linking experiments with RNA polymerase IV, and intact transcription factors. After binding the proteins to the labeled DNA, proteins that are close to the BrdU residues in the two boxes can be cross-linked by UV irradiation. Then the unbound proteins can be washed away, the DNA digested with DNase, and the remaining proteins subjected to SDS-PAGE. Those proteins (TBP and TAFs) that were bound to each of the promoter boxes will be cross-linked to labeled DNA and so will be detectable on the gel by autoradiography. These conclusions can be checked by repeating the photo-cross-linking experiments with TBP and only the TAFs that bound to each promoter box. These proteins should again be cross-linked in these experiments. To double-check the binding specificities, these same proteins can be tested for binding to each promoter box using DNase footprinting. The TAFs that were cross-linked to each box should also make a footprint in the same box. 4. Phosphorylation of serines 2 and 5 of the CTD of the Rpb1 subunit of RNA polymerase II by the protein kinase of TFIIH is required for initiation of transcription. Thus, an inhibitor of this protein kinase will block initiation, including the first melting of the DNA at the promoter, and formation of abortive transcripts. The in vitro transcription experiments would therefore show essentially no transcription with the inhibitor, but at least abortive transcription in the absence of the inhibitor. Because RNA polymerase II would still bind to the promoter, the DNase footprinting experiments would show a footprint in the presence of the inhibitor, but it would probably be a weak footprint because no open promoter complex would form. Furthermore, with no promoter melting, the footprint would show no local enhancement of DNase cleavage, such as that revealed by the dark band at about position -40 in Figure 11.12. The footprint in the presence of the inhibitor would probably also be smaller, not only because of the weaker binding of polymerase to the promoter, but because no abortive transcripts would be made. Without the inhibitor, abortive transcripts up to 12 nt long are made, and that would presumably require the polymerase to move a few base pairs downstream, enlarging the footprint. 5. Using site-directed mutagenesis, introduce cysteine residues into domains of protein X that you suspect interact with protein Y. To each of these cysteines in turn, attach the iron-EDTA complex Fe-BABE. Next, bind the two proteins together, and activate hydroxyl radical production by Fe-BABE. If a cysteine residue is within 15 of a site in protein Y in the complex, this site will be cleaved by the hydroxyl radical. Next, subject the proteins to SDS-PAGE, Western blotting, and probing with an antiserum that reacts with multiple sites on protein Y. A band of reduced size, not seen in control experiments without hydroxyl radical production, indicates that a particular cysteine in protein X makes close contact with protein Y, and is therefore in a domain that is probably involved in protein-protein interaction.

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