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. 1997 Mar 4;94(5):1733-8.
doi: 10.1073/pnas.94.5.1733.

Cloning of murine RNA polymerase I-specific TAF factors: conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1

J Heix  1 , J C Zomerdijk, A Ravanpay, R Tjian, I Grummt
Affiliations

Cloning of murine RNA polymerase I-specific TAF factors: conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1

J Heix et al. Proc Natl Acad Sci U S A. .

Abstract

Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs). Unlike class II- and III-specific TBP-TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter. As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAFIs) and compared the amino acid sequences of the murine TAFIs with their human counterparts. The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAFIs, demonstrating that differences in the primary structure of human and mouse TAFIs do not dramatically alter the network of protein-protein contacts responsible for assembly of the multimeric complex. Thus, primate vs. rodent promoter selectivity mediated by the TBP-TAFI complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription.

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Figures

Figure 1
Figure 1
Alignment of the deduced amino acid sequences of murine and human TAFIs. The sequence of murine TAFIs is shown in the upper line; the sequence of human TAFIs is shown in the lower line. Identical amino acids are boxed, and gaps introduced for best alignment are indicated by hyphens.
Figure 2
Figure 2
Characterization of the recombinant TAFIs. (A) Recombinant TAFIs are indistinguishable from the endogenous subunits of TIF-IB. Recombinant mTAFIs and TIF-IB were analyzed by immunoblotting using antisera against the individual TAFIs as indicated. (B) Antibodies against mTAFI95 precipitate a protein complex containing TBP. Nuclear extract was incubated with immobilized IgGs from preimmune serum (lanes 1–3) or α-mTAFI95 serum (lanes 4–6). The immunoprecipitates were analyzed by immunoblotting using αTBP antibodies (mAb 3G3). (C) α-mTAFI95 antibodies deplete TIF-IB activity from nuclear extract. Transcription was assayed in untreated extract (lanes 1 and 4) or in extract treated with bead-bound preimmune (lanes 2 and 5) or α-mTAFI95 serum (lanes 3 and 6) either in the absence of additional factors (lanes 1–3) or in the presence of immunopurified TIF-IB (lanes 4–6). (D) TIF-IB precipitated by α-mTAFI95 antibodies is transcriptionally active. Nuclear extract was incubated with bead-bound preimmune (lanes 4–6) or α-mTAFI95 serum (lanes 7–9), and aliquots of the suspended beads (1, 3, and 5 μl) were assayed for TIF-IB activity in a reconstituted transcription system. Transcripts synthesized after addition of 40 and 80 pg of immunopurified TIF-IB are shown in lanes 2 and 3.
Figure 3
Figure 3
TAFI–TBP and TAFI–TAFI interactions are conserved between mouse and human. GST and GST–mTBP were immobilized on glutathione–Sepharose and incubated with [35S]Met-labeled TAFIs. FLAG epitope-tagged mouse TAFIs were immobilized on M2 antibody beads directed against the FLAG epitope and incubated with [35S]Met-labeled TAFIs or TBP. As a control, the antibody beads preincubated with crude Sf9 cell extract were used. Bound complexes were analyzed by SDS/PAGE and autoradiography. The “load” shows 10% of the input proteins.
Figure 4
Figure 4
Assembly of chimeric TIF-IB/SL1 complexes from recombinant subunits in vitro. Recombinant TBP and TAFIs were combined as indicated and assembled into TBP–TAFI complexes. Complexes were immunopurified with α-FLAG antibodies, eluted with the epitope peptide, and analyzed on a silver-stained SDS/polyacrylamide gel.
Figure 5
Figure 5
Recombinant TBP–mTAFI complexes do not reconstitute TIF-IB activity. (A) Affinity-purified TIF-IB, but not recombinant TBP–TAFI complexes, is transcriptionally active. Lanes: 1, transcriptional activity in the absence of TIF-IB; 2–5, transcriptional activity in the presence of picogram amounts of immunopurified cellular TIF-IB; 6 and 7, the reactions that have been complemented by addition of 100 pg of the recombinant TBP–mTAFI complexes shown in B. The recombinant complexes also were inactive if added at higher or lower amounts (not shown). (B) Subunit composition of cellular TIF-IB and recombinant TBP–TAFI complexes. Silver-stained SDS/polyacrylamide gels showing the subunits of cellular TIF-IB (lane 1), recombinant TBP–mTAFI complexes assembled from purified subunits (lane 2), and complexes assembled in baculovirus-infected Sf9 cells (lane 3). The individual complexes contained differently tagged subunits (see Materials and Methods) and were analyzed on separate gels. Therefore, the electrophoretic mobility of the subunits is not identical.
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