PARA DETERMINAR EL NIVEL DE CONOCIMIENTO DE LA MADRES

The multistep model or the stepwise model for initiation complex formation has been called into question by the biochemical identification of complexes termed holoenzymes. The complexes contain Pol II, a subset of basal factors, SRB proteins (suppressors of Pol II CTD truncations) and other components (see below).

1.1.2.1 General requirement for SRB components in vivo

The SRB proteins were identified in a genetic screen for CTD-interacting polypeptides and were isolated as suppressors of CTD-truncation phenotype (Nonet and Young, 1989). A number of the SRB genes are essential for yeast cell growth (Koleske et al., 1992; Thompson et al., 1993). While the SRB proteins are not

necessary for in vitro transcription in minimal systems reconstituted with purified

GTFs, at least a subset of SRB proteins are essential for in vitro transcription using

nuclear extracts. Moreover, temperature-sensitive mutations in the SRB4 and SRB6

genes affect transcription of all genes examined so far (Koleske and Young, 1995).

Of the non-essential SRB genes SRB8^ SRB9, SRB 10 and SRB 11 are identical to

the SSN 5, SSN 2, SSN3 and SSN8 gene products respectively and belong to the SWI/SNF complex (Song et al., 1996). The SSN genes were isolated as suppressors of a yeast mutant lacking the Snfl protein kinase, which is required for glucose repression in yeast. The SSN genes are all non-essential^ and have been suggested to function in repression of many Pol II promoters.

1.1.2.2 Yeast Holoenzyme

Young and colleagues recently reported the presence of the first form of the holoenzyme that consists of Pol II, 9 SRBs, TFIIB, TFIIF, TFIIH, and other proteins. This complex is capable of accurate initiation when supplemented with TBP and TFIIE (Koleske and Young, 1994).

The holoenzyme contains only a portion of the Pol II found in a cellular extract. The holoenzyme was discovered through the use of antibodies against transcriptional regulatory proteins encoded by SRB genes. Quantitative westem-blot analysis revealed

that the SRB proteins accumulate to only 5-20% of the level of Pol II subunits in cell

extracts, and that most of the SRB protein is tightly complexed in 1-1 stoichiometery with this fraction of Pol II during holoenzyme purification (Koleske and Young, 1994). The fraction of Pol II associated with SRB proteins has been estimated to be as high as 50% (Kim et al., 1994). The lower estimates however, are consistent with immunoprécipitation experiments which show that the majority of Pol II molecules in

crude whole-cell extracts are not associated with other polypeptides (Kolodziej et al., 1990). Thus, the holoenzyme may have been m issed earlier in standard chromatographic approaches to Pol II purification using non-specific chain elongation assays, because it accounts for only fraction of the total Pol II activity.

1.1.2.3 Mediator

A second form of yeast Pol II holoenzyme has been purified by Kornberg's group that appears to be a sub complex of the original holoenzyme isolated by Young and colleagues. This m ultisubunit complex contains SRB proteins, TFIIF,

GAL1HSPT13, SU G l and approximately ten other proteins which can be dissociated from a preparation of Pol II holoenzyme by using antibodies against the CTD; this 'mediator' complex is necessary to reconstitute the response to activators (Kim et al.,

1994). These data support genetic studies suggestive of a role for G A L II and SUGl \n

mediating transcriptional activation (Koleske and Young, 1995). The data also confirm

observations that SRB2, SRB 5 and G A L II stimulate basal and activated transcription

when added back to nuclear extracts devoid of these activities (Koleske et al., 1992; Thompson et al., 1993). Thus transcriptional activators can potentially interact both with components of TFIID and with the holoenzyme. An important difference between the holoenzymes of the Young and the Komberg laboratories is the occurrence, in the former, of additional transcription proteins, including TFIIB, TFIIH and the SWI-SNF complex (Wilson et al., 1996).

1.1.2.4 Mammalian Holoenzyme

Recently, the mammalian holoenzyme complex was purified by two independent groups. Schibler's group isolated the holoenzyme by immunoprécipitation of the M015/CDK7 subunit of TFIIH from rat liver nuclear extracts (Ossipow et al.,

1995). Their holoenzyme contains all of the components necessary for transcription initiation including certain TAFs, with exception of TBP and TFIIB, as well as human homologs of several SRBs and DNA repair proteins but is unresponsive to transcriptional activators. While Reinberg's group used an antibody against the largest subunit of TFIIF (RAP74) to purify the holoenzyme from HeLa nuclear extracts (Maldonado et al., 1996). This holoenzyme was composed of a subset of GTFs (core

Pol II, TFIIE, TFIIF, and TFIIH), human SRBs (human SRB7, SRBIO, S R B ll), and

proteins involved in nucleotide repair (DNA Pol £, RPA, RFC and HRAD-51). In addition, the holoenzyme contained mediator proteins (Kim et al., 1994; see below), which bound to the CTD and conferred responsiveness to the acidic activator VP 16.

From the studies described thus far, it is clear that a number of RNA Pol II complexes have been isolated containing different subsets of the GTFs in yeast and in mammalian cells. Adding complexity to the RNA Pol II holoenzyme complexes are the findings that the mammalian holoenzyme contain a large num ber of other polypeptides, some of which may play important roles in nucleotide excision repair, DNA double strand break and or cell cycle check point control (Maldonado et al.,

1996) and chromatin remodelling (Wilson et al., 1996).

1.1.2.5 Holoenzymes and activators

pot'-j f f ai-.

The observation by Young and colleagues that only a fraction of the ce llu la ^ s present in the holoenzyme initially raised doubts about its authenticity. However, because almost all of the SRBs are associated, the finding that a temperature-sensitive

SRB4 m utant leads to a complete shut-off of mRNA synthesis at the restrictive tem perature provides a com pelling argum ent that the holoenzym e is the transcriptionally active form of RNA polymerase in vivo. In further support of the model, purified mammalian Pol II, TFIIF, TFIIB and TFIIH can also form a complex independently of DNA template in vitro (Serizawa et al., 1993). The presence of holoenzyme in eukaryotic cells has important implications for the mechanism of gene activation, perhaps best considered by using E.coli holoenzyme as a model system (Figure 1.3).

The E.coli RNA polymerase holoenzyme consists of a four-subunit core (a2pp') complexed with a o subunit. Promoter recognition is conferred by the C-terminus of the a subunit, which in some promoters binds the 'UP' element at position -50 (Ross et al., 1993), and a a factor, often which binds the conserved -35 and -10 regions. Prokaryotic activators stimulate transcription in a single step by contacting either the a subunit, in the case of the catabolite activator protein (CAP) (Chen et al., 1994), or the G subunit, the case of the bacteriophage X cl protein (Li et al., 1994). These interactions have been shown to enhance transcription by increased recruitment of the holoenzyme to the promoter, or isomérisation from the closed to the open complex. W ithin an artificial promoter context, CAP and cl evidently bind the holoenzyme simultaneously, generating a synergistic response (Joung et al., 1994).

Figure 1.3. Model for assembly of the RNA polymerase II (Pol II) holoenzyme into an initiation complex.

Activators (ACT) function by contacting components of the holoenzymes in

prokaryotes and {E.coli) and eukaryotes {S. cerevisiae). (A) In E.coli, putative

activator targets include the a and a subunits. (B) In yeast, putative targets include TBP, the mediator. General transcription factors such as TFIIB and TFIIH, and TAFs. The functional consequences of all these interactions would be the same: recruitment of the holoenzyme and/or isomérisation from an inactive to an active form (Adapted from Carey, 1995).

The Holoenzyme Model