Assembly and isolation of intermediate steps of transcription complexes formed on the human 5S rRNA gene

Abstract

By employing purified transcription factors and RNA polymerase III (pol III), we generated active pol III transcription complexes on the human 5S rRNA gene. These large complexes were separated by size exclusion chromatography from non- incorporated proteins. In addition, we succeeded in isolating specific intermediate stages of complex formation. Such isolated partial complexes require complementation with the missing activities for full transcription activity. One central finding is that a 5S DNA-TFIIIA-TFIIIC2-TFIIIBbeta complex could be isolated which had been assembled in the absence of the general pol III transcription factor IIIC1. Thus TFIIIC1 is not an assembly factor for other transcription factors. Although pol III has the potential to bind unspecifically to DNA, such polymerase molecules cannot be rendered initiation competent by direct recruitment to a 5S DNA-TFIIIA-TFIIIC2- TFIIIBbeta complex, but this process strictly requires additional TFIIIC1 activity. This clearly demonstrates that in contrast to yeast cells, hTFIIIB(beta), although required, does not suffice for the functional recruitment of polymerase III. These data document that TFIIIC1 is the second transcription factor required for the recruitment of pol III in mammalian cells.

Figure 1

Isolation of active 5S transcription complexes from purified transcription factors and RNA polymerase III. (A) A standard in vitro transcription was performed using the following fractions: 2.5 µl of TFIIIA (protein concentration: 0.3 mg/ml), 2.5 µl of TFIIIBβ (0.4 mg/ml), 1 µl of pol III (0.1 mg/ml), 7.5 µl of TFIIIC1 (0.3 mg/ml) and 2.5 µl of TFIIIC2 (0.1 mg/ml). Lane 1, complete reaction; lanes 2–6, reactions without TFIIIBβ, pol III, TFIIIC1, TFIIIC2 or TFIIIA, respectively. (B) Complete transcription complexes were assembled using the same fractions and the same stoichiometry as in (A), isolated and tested for transcription activity as described in Materials and Methods. Lane 1, input; lanes 2–9, fractions 9–16 from the S500 column. (C) Western blot. S500 elution profile of TFIIIC2, TFIIIBβ and pol III not bound to DNA. (D) Western blot of the fractions stemming from the S500 column with 5S transcription complexes from (B).

Figure 2

Isolation of a ternary 5S–TFIIIA–TFIIIC2 complex. A 5 µl aliquot of TFIIIA and 2.5 µl of TFIIIC2 were pre-incubated with the 5S plasmid. The resulting complexes were isolated by S500 chromatography as described before. The input and the fractions from the column were complemented with TFIIIC1, TFIIIBβ and pol III in the same amounts as in Figure 1 and tested for transcription activity. Lane 1, input; lanes 2–10, S500 fractions 8–16.

Figure 3

Stable quaternary 5S–TFIIIA–TFIIIC2–TFIIIBβ can be isolated, but not a 5S–TFIIIA–TFIIIC2–TFIIIC1 complex. (A) The pre-incubation was performed with TFIIIA, TFIIIC2 and TFIIIC1. The input and the fractions from the S500 column were supplemented with activities as indicated. Lanes 1–2, input (I); lanes 3–10, S500 fractions 8–15, supplemented with TFIIIBβ and pol III; lanes 11–17, fraction 11 supplemented with different activities as indicated. (B) Pre-incubation with TFIIIA, TFIIIC2 and TFIIIBβ; prior to post-incubation, activities were supplemented as indicated. Lanes 1 and 2, input; lanes 3–10, S500 fractions 9–16 supplemented with TFIIIC1 and pol III; lanes 11–16, fraction 11 supplemented with different activities as indicated. (C) The 5S gene was incubated together with either TFIIIA and TFIIIBβ (first run, left panel, lanes 1–11), or TFIIIA, TFIIIC2 and TFIIIBβ (second run, right panel, lanes 12–14). After S500 chromatography, the resulting fractions were supplemented either with TFIIIC2, TFIIIC1 and pol III (left panel) or with TFIIIC1 and pol III (right panel). Where indicated, 2 or 4 µl of TFIIIBβ were added. Lane 1; input (run 1); lanes 2–8, S500 fractions 9–15 (run 1); lanes 9–11, fraction 11 (run 1) with 0, 2 and 4 µl of TFIIIBβ; lanes 12–14, fraction 11 (run 2) with 0, 2 and 4 µl of TFIIIBβ.

Figure 4

Recruitment of TFIIIC1 or polymerase III into the TFIIIA– TFIIIC2–TFIIIBβ complex. The procedure was similar to the previous experiments, with the following modifications. (A) Pre-incubation of the 5S gene with TFIIIA, TFIIIC2, TFIIIBβ and TFIIIC1. The input (I) (lanes 1 and 2) and the resulting fractions from the S500 chromatography (lanes 3–14) were supplemented with additional activities as indicated. (B) Pre-incubation of the 5S gene with TFIIIA, TFIIIC2, TFIIIBβ and pol III. The input (I) (lanes 1 and 2) and the resulting fractions from the S500 chromatography (lanes 3–14) were supplemented with additional activities as indicated.

Figure 5

Pre-incubation of 5S plasmid DNA with transcription factors TFIIIC2, TFIIIBβ, TFIIIC1 and pol III, and subsequent analysis of potential complexes. After S500 chromatography, the input (lanes 1–3) or the peak fraction 11 of the void volume (lanes 4–14) were incubated with 2.5 or 5 µl of TFIIIA, 2.5 µl of TFIIIC2, 2.5 µl of TFIIIBβ, 7.5 µl of TFIIIC1 or 1 µl of pol III as indicated.