SV40 large T antigen binds to the TBP-TAF(I) complex SL1 and coactivates ribosomal RNA transcription.
SV40 large T antigen is a multifunctional regulatory protein that plays a key role in the viral life cycle and can stimulate cell proliferation. To accomplish this, large T antigen has to control the expression of cellular genes involved in cell cycle progression and cell growth. rRNA synthesis by RNA polymerase I (Pol I) is tightly associated with cell growth and proliferation, and previous studies indicated that large T antigen up-regulates RNA Pol I transcription in SV40-infected cells. How this process occurs is currently unclear. To investigate the mechanisms of large T antigen stimulation of RNA Pol I transcription, we have established an in vitro transcription system that is responsive to large T antigen. Here, we show that recombinant large T antigen stimulates Pol I transcription reconstituted with purified RNA Pol I, UBF, and the TBP/TAF complex SL1. Immunoprecipitation experiments revealed that large T antigen directly binds to SL1, in vitro, as well as in SV40-infected cells. In addition, our data indicate that this interaction occurs by direct association with three SL1 subunits, namely TBP, TAF(I)48, and TAF(I)110. Transcription studies with large T antigen deletion mutants show that the 538-amino-acid amino-terminal domain is necessary for full stimulation of Pol I transcription. Importantly, mutants that no longer bind to SL1 are also unable to stimulate Pol I transcription. This indicates that recruitment of large T antigen to the rRNA promoter by SL1 constitutes a crucial step in the activation process. Taken together with recent studies on large T antigen activation of RNA Pol II transcription, these results suggest that viral modulation of genes involved in cell proliferation involves direct targeting of promoter-specific TBP/TAF complexes (i.e., SL1 or TFIID) by large T antigen.
Zhai
W
W
Department of Molecular Microbiology and Immunology, University of Southern California, School of Medicine, Los Angeles 90033, USA.
Tuan
J A
JA
Comai
L
L
D000952:Antigens, Polyomavirus Transforming / Q000302:isolation & purification / Q000378:metabolism*; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D002472:Cell Transformation, Viral; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005982:Glutathione Transferase; D006367:HeLa Cells; D006801:Humans; D035463:Pol1 Transcription Initiation Complex Proteins; D011485:Protein Binding; D012318:RNA Polymerase I / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013539:Simian virus 40 / Q000502:physiology*; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D035463:Pol1 Transcription Initiation Complex Proteins; D012335:RNA, Ribosomal; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C063660:transcription initiation factor TIF-IB; D005982:Glutathione Transferase; D012318:RNA Polymerase I