Is actin a transcription initiation factor for RNA ... - Europe PMC

The EMBO Joumal vol.3 no.lOpp.2363-2371, 1984

Is actin a transcription initiation factor for RNA polymerase B?

J.M.Egly, N.G.Miyamoto, V.Moncollin and P.Chambon Laboratoire de Genetique Moleculaire des Eucaryotes du CNRS, Unite 184 de Biologie Mol&ulaire et de Genie Genetique de l'INSERM, Institut de Chimie Biologique, Faculte de Medecine, 11, Rue Humann, 67085 Strasbourg Cedex, France Communicated by P.Chambon

We have previously reported that two fractions derived from HeLa cell S100 extracts, the heparin flow-through and the heparin 0.6 M KCI eluate are required in vitro for efficient and accurate transcription by RNA polymerase class B (II). We have further purified a factor present in the heparin flowthrough fraction, which markedly stimulates specific transcription catalyzed by the heparin 0.6 M KCI eluate. We report here that some of the properties of the stimulatory factor present in our most purified fractions are strikingly similar to those of actin. We demonstrate also that this factor acts at the pre-initiation level of the transcription reaction. Key words: RNA polymerase B/transcription/actin/initiation factor

Introduction The transcription of protein-coding genes in eucaryotic systems is performed by RNA polymerase B (II), which in vitro requires supplementation with crude cellular extracts to initiate accurate transcription (Weil et al., 1979; Manley et al., 1980) ['accurate transcription' refers here to initiation at the in vivo start (cap) sites]. Such cellular extracts, most notably prepared from HeLa cells and termed either S100 (Weil et al., 1979), whole cell extract (Manley et al., 1980) or nuclear extract (Dignam et al., 1983), contain multiple complementary fractions required to direct accurate transcription (for references, see Matsui et al., 1980; Tsai et al., 1981; Samuels et al., 1982). Some of these fractions contain factors which recognize specific promoter elements, such as the TATA box (see Breathnach and Chambon, 1981; Davison et al., 1983; Parker and Topol, 1984) and the 21-bp repeat upstream element of the SV40 promoter region (see Baty et al., 1984; Dynan and Tjian, 1983). We have reported that at least two factors present in fractions derived from HeLa cell S100 extracts, the heparin flowthrough (HFT) and the heparin 0.6 M KCl eluate (HO.6) (see Figure 1 and Davison et al., 1983), are involved in the TATA box-dependent formation of a stable pre-initiation complex with either the adenovirus-2 major late or the chicken conalbumin gene promoter. Our results suggested that a factor present in the HFT fraction was required for efficient formation of the stable complex. This paper reports the purification and characterization of a 43-kd stimulatory transcription factor present in the HFT. We also describe striking similarities between this transcription factor and actin, and its potential role at the pre-initiation level of RNA polymerase B-dependent specific transcription. ( IRL Press Limited, Oxford, England.

Results and Discussion Purification of the heparin-agarose flow-through (HFT) 43-kd stimulatory transcription factor The procedure used to fractionate HeLa cell S100 extract is shown schematically in Figure 1. As reported (Davison et al., 1983), both the HFT and the 0.6 M KCl eluate (HO.6) from a heparin-Ultrogel column are required for efficient and accurate in vitro synthesis of the adenovirus-2 major late 560 nucleotide run-off transcript (Figure 2A, lane 1). We describe below (see Table I for a summary) a fractionation procedure aimed at purifying the stimulatory transcription factor (STF) present in the HFT. The same procedure is also applicable to HFT fractions obtained from either HeLa whole cell extracts (not shown) or calf thymus S100 extracts. In an attempt to quantitate our results, we have defined one unit of STF as the amount of stimulatory activity which is required for maximal run-off transcription under the standard assay conditions described in Materials and methods. Consequently, the STF specific activity of any particular fraction is expressed as the inverse of the amount of protein necessary for maximal transcription under these standard conditions. We are aware of the serious limitations inherent in this measurement of STF activity, since at the present stage, specific in vitro transcription is the result of the interaction of many components in a complex reaction mixture HeLa cell S-100 extract

I

Heai.Utoe

0.66M

KC1 eluate ( H0. 6 ]

O.1M KCI flow-through

(HFT] I

4EAE-cellulose 0.35M KCI eluate

[DEO.35]

Cibacron-Blue Agarose

O.11M

KC1

salt-elution

fl ow-throug h

0.11M

KCI

flow-through [CBFT] kA34-Ul trogel I

I 0. 3 15M KC1 [ P'0.35]

~

0.55M KC1 [P0.5]

-

-I 1M KCI

[P1.0]

Void volume

(AcAIVV] Depolymeri sation

kA34-Ultrogel II 43 Kd Protein

[AcAI IP43]

Fig. 1. Scheme for the fractionation of RNA polymerase B transcription factors (see Materials and methods, Table I and text).

2363

J.M.Egly et al.

(see below for additional difficulties in the quantitation of our results). DEAE-cellulose chromatography. The HFT was applied directly onto a DEAE-cellulose column equilibrated in buffer A. As previously shown (Davison et al., 1983), the 0.35 M KC1 fraction (DEO.35) contained the STF activity (Figure 2A and B, compare lanes C, 1 and 2). Approximately 60700o of the protein present in the HFT was eliminated with little change in STF specific activity (Figure 3 and Table I). Cibacron Blue-agarose chromatography. After overnight dialysis against buffer A containing 0.05 M KC1, the DEO.35 was adsorbed onto a Cibacron Blue-agarose column (Materials and methods). Under these conditions, the Cibacron Blue flow-through fraction (CBFI) contained, at least in the case of calf thymus, the STF activity (Figure 2A and B, lanes 3). More than 707o of the total protein was found to be retained on the column (Figure 3, Table I). We always noted an important apparent loss of activity at this step. In particular, the HeLa cell CBFT was observed to be significantly less active than either the DEO.35 or HFT from which it was derived, although the decrease in activity was not usually as striking as shown in Figure 2A, lane 3. An explanation for this is discussed below. AcA34-Ultrogel chromatography I and II. The CBFT was precipitated with ammonium sulfate, dialyzed against buffer A containing 0.05 M KCI and loaded onto an AcA34-Ultrogel column equilibrated in the same buffer (Materials and methods). Figure 4A and B shows an SDS-polyacrylamide gel profile of the protein separation achieved in the case of HeLa cells and the STF activity of the corresponding fractions (see also Table I). The STF activity was not eluted as a discrete peak, but was present in almost all the fractions (Figure 4 and Table I). A 43-kd mol. wt. polypeptide which could be detected in almost all of these fractions appeared to be extensively purified in the void volume (VV), where it represented a significant fraction of the total 43-kd polypeptide present in the CBFT (Figures 3 and 4 and Table I). To determine if the chromatographic behaviour of the 43-kd polypeptide on AcA34-Ultrogel was due to an aggre-

gation phenomenon, the CBFT

was dialyzed against a depolymerization buffer (Materials and methods) prior to loading onto an AcA34-Ultrogel column equilibrated in the same buffer. Under these conditions, most of the 43-kd polypeptide was eluted in the 43-kd protein size region of the AcA34Ultrogel column (Figure 4C). The STF activity was also shifted to the same region (Figure 4D). These observations prompted us to pool the void volume region (AcAIVV) from the first AcA34-Ultrogel column (Figure 4A) and to dialyze it against the depolymerization buffer prior to loading onto a second AcA34-Ultrogel column equilibrated in the same buffer. Under these conditions, we observed a displacement of the 43-kd polypeptide from the A

Hela Cells

B

Calf Thymus

cl

> o

C

V)

L--

+

LO)

+.

>

I

0