Tuesday, 21 April 2015

EPITOPE TAGGING METHOD FOR RNA PLYMERASE 2: Principle procedure & applications of epitope tagging method.

RNA Polymerase Subunit Structures 

The first subunit structures for a eukaryotic RNA polymerase (polymerase II) were reported independently by Pierre Chambon and Rutter and their colleagues in 1971, but they were incomplete. We should note in passing that Chambon named his three polymerases A, B, and C, instead of I, II, and III, respectively. However, the I, II, III nomenclature of Roeder and Rutter has become the standard. We now have very good structural information on all three polymerases from a variety of eukaryotes. The structures of all three polymerases are quite complex, with 14, 12, and 17 subunits in polymerases I, II, and III, respectively. Polymerase II is by far the best studied, and we will focus the rest of our discussion on the structure and function of that enzyme.



Polymerase II Structure  

For enzymes as complex as the eukaryotic RNA polymerases it is difficult to tell which polypeptides that co purify with the polymerase activity are really subunits of the enzymes and which are merely contaminants that bind tightly to the enzymes. One way of dealing with this problem would be to separate the putative subunits of a polymerase and then see which polypeptides are really required to reconstitute polymerase activity. Although this strategy worked beautifully for the prokaryotic polymerases, no one has yet been able to reconstitute a eukaryotic nuclear polymerase from its separate subunits. Thus, one must try a different tack. Another way of approaching this problem is to fi nd the genes for all the putative subunits of a polymerase, mutate them, and determine which are required for activity. This has been accomplished for one enzyme: polymerase II of baker’s yeast, Saccharomyces cerevisiae. Several investigators used traditional methods to purify yeast polymerase II to homogeneity and identified 10 putative subunits. Later, some of the same scientists discovered two other subunits that had been hidden in the earlier analyses, so the current concept of the structure of yeast polymerase II includes 12 subunits. The genes for all 12 subunits have been sequenced, which tells us the amino acid sequences of their products. The genes have also been systematically mutated, and the effects of these  mutations on polymerase II activity have been observed.


How do the structures of polymerases I and III compare with this polymerase II structure? First, all the polymerase structures are complex—even more so than the structures of the bacterial polymerases. Second, all the structures are similar in that each contains two large (greater than 100 kD) subunits, plus a variety of smaller subunits. In this respect, these structures resemble those of the prokaryotic core polymerases, which contain two high-molecular-mass subunits (b and b9) plus three low-molecular-mass subunits (two a’s and an v). In fact, as we will see later in this chapter, an evolutionary relationship is evident between three of the prokaryotic core polymerase subunits and three of the subunits of all of the eukaryotic polymerases. In other words, the three eukaryotic polymerases are related to the prokaryotic polymerase and to one another.


Richard Young and his coworkers originally identified 10 polypeptides that are authentic polymerase II subunits, or at least tightly bound contaminants. The method they used is called epitope tagging, in which they attached a small foreign epitope to one of the yeast polymerase II subunits (Rpb3) by engineering its gene. Then they introduced this gene into yeast cells lacking a functional Rpb3 gene, labeled the cellular proteins with either 35S or 32P, and used an antibody directed against the foreign epitope to precipitate the whole enzyme. After immuno precipitation, they separated the labeled polypeptides of the precipitated protein by SDS-PAGE and detected them by autoradiography. Figure presents the results. This single-step purification method yielded essentially pure polymerase II with 10 apparent subunits. We can also see a few minor polypeptides, but they are equally visible in the control in which wild-type enzyme, with no epitope tag, was used. Therefore, they are not polymeraseassociated. Figure shows a later SDS-PAGE analysis of the same polymerase, performed by Roger Kornberg and colleagues, which  distinguished 12 subunits. Rpb11 had coelectrophoresed with Rpb9, and Rpb12 had coelectrophoresed with Rpb10, so both Rpb11 and Rpb12 had been missed in the earlier  experiments.


Principle of epitope tagging. 

An extra domain (an epitope tag, red) has been added genetically to one subunit (Rpb3) of the yeast RNA polymerase II. All the other subunits are normal, and assemble with the altered Rpb3 subunit to form an active polymerase. This polymerase has also been labeled by growing cells in labeled amino acids. (a) Add an antibody directed against the epitope tag, which immunoprecipitates the whole RNA polymerase, separating it from contaminating proteins (gray). This gives very pure polymerase in just one step. (b) Add the strong detergent SDS, which separates and denatures the subunits of the purifi ed polymerase. (c) Electrophorese the denatured subunits of the polymerase to yield the electropherogram at bottom.








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