Identification of the Target of a Transcription Activator Protein by Protein-Protein Photocrosslinking

Chen, Yan; Ebright, Yon W.

doi:10.1126/science.8016656

Cited by 169 publications

(118 citation statements)

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“…The single strand binding protein of phage N4 called N4SSB contacts the ␤Ј subunit (52), whereas repressor contacts the carboxyl terminus of the ␣ subunit of RNAP. Although mutations at the contact points of either the activator or RNA polymerase can cause loss of transcription activation, the loss of one contact can be relieved by the establishment of a different contact with a different subunit of RNAP (51)(52)(53)(54). Thus, these interactions, although biologically important, need not be very specific.…”

Section: Discussionmentioning

confidence: 99%

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Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Mohanty

Sahoo

Bastia

1998

Journal of Biological Chemistry

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Since DNA replication and transcription often temporally and spatially overlap each other, the impact of one process on the other is of considerable interest. We have reported previously that transcription is impeded at the replication termini of Escherichia coli and Bacillus subtilis in a polar mode and that, when transcription is allowed to invade a replication terminus from the permissive direction, arrest of replication fork at the terminus is abrogated. In the present report, we have addressed four significant questions pertaining to the mechanism of transcription impedance by the replication terminator proteins. Is transcription arrested at the replication terminus or does RNA polymerase dissociate from the DNA causing authentic transcription termination? How does transcription cause abrogation of replication fork arrest at the terminus? Are the points of arrest of the replication fork and transcription the same or are these different? Are eukaryotic RNA polymerases also arrested at prokaryotic replication termini? Our results show that replication terminator proteins of E. coli and B. subtilis arrest but do not terminate transcription. Passage of an RNA transcript through the replication terminus causes the dissociation of the terminator protein from the terminus DNA, thus causing abrogation of replication fork arrest. DNA and RNA chain elongation are arrested at different locations on the terminator sites. Finally, although bacterial replication terminator proteins blocked yeast RNA polymerases in a polar fashion, a yeast transcription terminator protein (Reb1p) was unable to block T7 RNA polymerase and E. coli DnaB helicase.The chromosomes of Escherichia coli and Bacillus subtilis, and some plasmids like R6K, initiate replication from specific origins and the replication forks moving either bi-or unidirectionally, are arrested at sequence-specific replication termini (1-3). Sequence-specific replication fork barriers have also been observed in the nontranscribed spacer regions of ribosomal DNAs of Saccharomyces cerevisiae (4, 5), plants (6), Xenopus (7), and human (8).The replication termini are sequence-specific and bind to cognate terminator proteins, and the protein-DNA complex arrests replication forks in an orientation-dependent manner (9, 10). DnaB (11,12,19). Detailed biochemical and biophysical analyses have been made possible in both the systems by the determination of the crystal structures of RTP at 2.6 Å (21) and of Tus-Ter () complex at 2.7 Å (22). The DNA binding (23), dimer-dimer interaction (24), and DnaB interaction domains (25) of RTP have been determined by genetic and biochemical analyses that used the crystal structure as a guide. Similarly, the DNA binding domain of Tus has been determined with the help of crystal structure and genetic analysis (22).The impact of transcription on the initiation (26, 27) and elongation stages of DNA replication have been reported (28,29). We have reported previously that Tus and RTP can block RNA chain elongation catalyzed by several prokaryotic RN...

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Section: Discussionmentioning

confidence: 99%

“…Preliminary work shows that the Y33A mutant of RTP fails to arrest RNAPs. 4 If protein-protein interaction is involved, future work will also be directed toward mapping physically the contact points between RNAPs and Tus and RTP by photo-cross-linking procedures (52,54 …”

Section: Discussionmentioning

confidence: 99%

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Mohanty

Sahoo

Bastia

1998

Journal of Biological Chemistry

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“…N-(-2-pyridyldithio)ethyl-4-azidosalicylamide (PEAS) is a heterobifunctional photo-reactive crosslinker (12). Radiolabeling of PEAS was identical to that described for APDP (10,11).…”

Section: Methodsmentioning

confidence: 99%

“…The denatured Gal4 derivative was refolded and then conjugated to another photoinducible crosslinker, PEAS. This crosslinker has a shorter spacer between the photo-reactive and cysteine-reactive functional groups (15 vs. 20 Å for APDP) (12). 125 I-PEAS was conjugated to the cysteine at position 856 in Gal4 [(1-100) ϩ (840-881)], and the experiment was performed as in Fig.…”

Section: Interactions In Vitromentioning

confidence: 99%

Transcriptional activating regions target a cyclin-dependent kinase

Ansari

Koh

Zaman

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Several yeast activators are phosphorylated by SRB10, a cyclindependent kinase associated with the transcriptional machinery. Sites of phosphorylation are found outside the activating region in each case, and the modification has different physiological consequences in different cases. We show here that certain acidic transcriptional activating regions contact SRB10 as assayed both in vivo and in vitro. The interaction evidently positions each activator, as it activates transcription, so that it gets phosphorylated by SRB10, and thus a common mechanism targets disparate substrates to the kinase. S everal yeast transcriptional activators become phosphorylated as they activate transcription (1-3). Phosphorylation has various consequences: it affects nuclear export of Msn2, degradation of Gcn4, inducer-dependent activation by Gal4, and the activity of Sip4 (1-3). In each case, the activator is phosphorylated on residues positioned outside its activating and DNA binding regions. For example, the most important sites phosphorylated in Gal4 (S690, S696, and S699) lie in the region between the DNA binding domain and the principal activating region (2). Nevertheless, under physiological conditions, both the DNA binding and the activating regions are required for these phosphorylations, just as they are required for activity of the activator (2, 4).The kinase believed responsible for the phosphorylations described above is the cyclin-dependent kinase SRB10 complexed with the cyclin SRB11 (1-3). The SRB10͞11 pair is part of a larger complex that includes SRB8 and SRB9, and that complex can, by some reports, associate with the RNA polymerase II mediator (5, 6). SRB10͞11 is also believed to phosphorylate other substrates in the transcriptional machinery (5-7). How the various transcriptional activators are targeted to the SRB10 kinase has heretofore been unknown. Here, we show that the activating region of Gal4 contacts SRB10 as measured in binding assays performed in vitro and in vivo. Binding experiments performed in vitro indicate that other acidic activating regions also interact with SRB10. Materials and MethodsProteins. Purifications of recombinant CDK͞cyclins were performed as described (6,8). Yeast RNA polymerase II holoenzyme was purified as in ref. 9. GST-Gal4wt (840-874) and GST-Gal4mut (840-874) (which contains the mutations V864E and L868V) were purified as in ref. 10. TATA binding protein (TBP), Gal80, and other Gal4 derivatives were purified as described (10, 11).For Fig. 1B, Gal4 derivative bearing the F856C substitution was overexpressed in BL21 (DE3) pLysS bacterial strain. Cells were lysed in buffer A (20 mM Hepes, pH 7.5͞150 mM KCl͞ 0.1% Nonidet P-40͞20 M ZnCl 2 ͞3 mM DTT͞1 mM ␤-mercaptoethanol͞10% glycerol͞1 mM PMSF͞0.5 mM benzamidine) containing 6 M urea. After centrifugation (15 min at 10,000 ϫ g), the protein was purified by using a SP-Sepharose fast flow (Amersham Pharmacia) column under denaturing conditions. The relevant fractions were concentrated and refolded by rapid dilution into buffer A with...

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“…Because this genetic approach proved difficult, we directly identified polypeptides near this surface by using site-specific photocross-linking. In this strategy, TFIIA was engineered to contain unique surface accessible cysteine residues near the TFIIA functional surface, and these residues were used to attach the 125 I-labeled photocross-linker PEAS, which has a probe length of 16Å (Chen et al 1994). PICs containing this modified recombinant TFIIA were isolated and treated with UV light to activate the crosslinker.…”

Section: Site-specific Photocross-linking Of Pics Identifies Three Pomentioning

confidence: 99%

Positive and negative functions of the SAGA complex mediated through interaction of Spt8 with TBP and the N-terminal domain of TFIIA

Warfield

Ranish²,

Hahn³

2004

Genes Dev.

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A surface that is required for rapid formation of preinitiation complexes (PICs) was identified on the N-terminal domain (NTD) of the RNA Pol II general transcription factor TFIIA. Site-specific photocross-linkers and tethered protein cleavage reagents positioned on the NTD of TFIIA and assembled in PICs identified the SAGA subunit Spt8 and the TFIID subunit Taf4 as located near this surface. In agreement with these findings, mutations in Spt8 and the TFIIA NTD interact genetically. Using purified proteins, it was found that TFIIA and Spt8 do not stably bind to each other, but rather both compete for binding to TBP. Consistent with this competition, Spt8 inhibits the binding of SAGA to PICs in the absence of activator. In the presence of activator, Spt8 enhances transcription in vitro, and the positive function of the TFIIA NTD is largely mediated through Spt8. Our results suggest a mechanism for the previously observed positive and negative effects of Spt8 on transcription observed in vivo.[Keywords: Transcription; SAGA; TFIIA; coactivator; repression] Supplemental material is available at http://www.genesdev.org.

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Identification of the Target of a Transcription Activator Protein by Protein-Protein Photocrosslinking

Cited by 169 publications

References 23 publications

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Transcriptional activating regions target a cyclin-dependent kinase

Positive and negative functions of the SAGA complex mediated through interaction of Spt8 with TBP and the N-terminal domain of TFIIA

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