Transcription Factor B Contacts Promoter DNA Near the Transcription Start Site of the Archaeal Transcription Initiation Complex

TY - JOUR

T1 - Transcription Factor B Contacts Promoter DNA Near the Transcription Start Site of the Archaeal Transcription Initiation Complex

AU - Renfrow, Matthew B.

AU - Naryshkin, Nikolai

AU - Lewis, L. Michelle

AU - Chen, Hung Ta

AU - Ebright, Richard H.

AU - Scott, Robert A.

PY - 2004/1/23

Y1 - 2004/1/23

N2 - Transcription initiation in all three domains of life requires the assembly of large multiprotein complexes at DNA promoters before RNA polymerase (RNAP)-catalyzed transcript synthesis. Core RNAP subunits show homology among the three domains of life, and recent structural information supports this homology. General transcription factors are required for productive transcription initiation complex formation. The archaeal general transcription factors TATA-element-binding protein (TBP), which mediates promoter recognition, and transcription factor B (TFB), which mediates recruitment of RNAP, show extensive homology to eukaryal TBP and TFIIB. Crystallographic information is becoming available for fragments of transcription initiation complexes (e.g. RNAP, TBP-TFB-DNA, TBP-TFIIB-DNA), but understanding the molecular topography of complete initiation complexes still requires biochemical and biophysical characterization of protein-protein and protein-DNA interactions. In published work, systematic site-specific protein-DNA photocrosslinking has been used to define positions of RNAP subunits and general transcription factors in bacterial and eukaryal initiation complexes. In this work, we have used systematic site-specific protein-DNA photocrosslinking to define positions of RNAP subunits and general transcription factors in an archaeal initiation complex. Employing a set of 41 derivatized DNA fragments, each having a phenyl azide photoactivable crosslinking agent incorporated at a single, defined site within positions -40 to +1 of the gdh promoter of the hyperthermophilic marine archaea, Pyrococcus furiosus (Pf), we have determined the locations of PfRNAP subunits PfTBP and PfTFB relative to promoter DNA. The resulting topographical information supports the striking homology with the eukaryal initiation complex and permits one major new conclusion, which is that PfTFB interacts with promoter DNA not only in the TATA-element region but also in the transcription-bubble region, near the transcription start site. Comparison with crystallographic information implicates the PfTFB N-terminal domain in the interaction with the transcription-bubble region. The results are discussed in relation to the known effects of substitutions in the TFB and TFIIB N-terminal domains on transcription initiation and transcription start-site selection.

AB - Transcription initiation in all three domains of life requires the assembly of large multiprotein complexes at DNA promoters before RNA polymerase (RNAP)-catalyzed transcript synthesis. Core RNAP subunits show homology among the three domains of life, and recent structural information supports this homology. General transcription factors are required for productive transcription initiation complex formation. The archaeal general transcription factors TATA-element-binding protein (TBP), which mediates promoter recognition, and transcription factor B (TFB), which mediates recruitment of RNAP, show extensive homology to eukaryal TBP and TFIIB. Crystallographic information is becoming available for fragments of transcription initiation complexes (e.g. RNAP, TBP-TFB-DNA, TBP-TFIIB-DNA), but understanding the molecular topography of complete initiation complexes still requires biochemical and biophysical characterization of protein-protein and protein-DNA interactions. In published work, systematic site-specific protein-DNA photocrosslinking has been used to define positions of RNAP subunits and general transcription factors in bacterial and eukaryal initiation complexes. In this work, we have used systematic site-specific protein-DNA photocrosslinking to define positions of RNAP subunits and general transcription factors in an archaeal initiation complex. Employing a set of 41 derivatized DNA fragments, each having a phenyl azide photoactivable crosslinking agent incorporated at a single, defined site within positions -40 to +1 of the gdh promoter of the hyperthermophilic marine archaea, Pyrococcus furiosus (Pf), we have determined the locations of PfRNAP subunits PfTBP and PfTFB relative to promoter DNA. The resulting topographical information supports the striking homology with the eukaryal initiation complex and permits one major new conclusion, which is that PfTFB interacts with promoter DNA not only in the TATA-element region but also in the transcription-bubble region, near the transcription start site. Comparison with crystallographic information implicates the PfTFB N-terminal domain in the interaction with the transcription-bubble region. The results are discussed in relation to the known effects of substitutions in the TFB and TFIIB N-terminal domains on transcription initiation and transcription start-site selection.

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U2 - 10.1074/jbc.M311433200

DO - 10.1074/jbc.M311433200

M3 - Article

C2 - 14597623

SN - 0021-9258

VL - 279

SP - 2825

EP - 2831

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 4

ER -