M EXPERIMENTAL FIGURE 11-4 Column chromatography separates and identifies the three eukaryotic RNA polymerases, each with its own sensitivity to a-amanitin. A

protein extract from the nuclei of cultured eukaryotic cells Is passed through a DEAE Sephadex column and adsorbed protein eluted (black curve) with a solution of constantly increasing NaCl concentration. Three fractions from the eluate subsequently showed RNA polymerase activity (red curve). At a concentration of 1 ^g/ml, a-amanitin inhibits polymerase II activity but has no effect on polymerases I and III (green shading). Polymerase III is inhibited by 10 ^g/ml of a-amanitin, whereas polymerase I is unaffected even at this higher concentration. [See R. G. Roeder, 1974, J. Biol. Chem. 249:241.]

subunits and 10-14 smaller subunits, some of which are present in two or all three of the polymerases. The best-characterized eukaryotic RNA polymerases are from the yeast S. cerevisiae. Each of the yeast genes encoding the polymerase subunits has been cloned and sequenced and the effects of gene-knockout mutations have been characterized. In addition, the three-dimensional structure of yeast RNA

polymerase II missing two nonessential subunits has been determined (see Figure 11-5). The three nuclear RNA polymerases from all eukaryotes so far examined are very similar to those of yeast.

The two large subunits (RPB1 and RPB2) of all three eukaryotic RNA polymerases are related to each other and are similar to the E. coli P' and p subunits, respectively

▲ FIGURE 11-5 Comparison of three-dimensional structures of bacterial and eukaryotic RNA polymerases. These Ca trace models are based on x-ray crystallographic analysis of RNA polymerase from the bacterium T aquaticus and RNA polymerase II from S. cerevisiae. (a) The five subunits of the bacterial enzyme are distinguished by color. Only the N-terminal domains of the a subunits are included in this model. (b) Ten of the twelve subunits constituting yeast RNA polymerase II are shown in this model.

Subunits that are similar In conformation to those in the bacterial enzyme are shown in the same colors. The C-terminal domain of the large subunit RPB1 was not observed in the crystal structure, but it is known to extend from the position marked with a red arrow. (RPB is the abbreviation for "R NA polymerase B," which is an alternative way of referring to RNA polymerase II.) [Part (a) based on crystal structures from G. Zhang et al.,1999, Cell 98:811. Part (b) from P Cramer et al., 2001, Science 292:1863.]

E. coli core RNA polymerase (a2ßß'ra) oll loll W ra"!

Eukaryotic RNA polymerases I II III

ß-like subunits

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