Additional enzyme-specific subunits

Additional enzyme-specific subunits

▲ FIGURE 11-6 Schematic representation of the subunit structure of the E. coli RNA core polymerase and yeast nuclear RNA polymerases. All three yeast polymerases have five core subunits homologous to the p, p', two a, and ra subunits of E. coli RNA polymerase. The largest subunit (RPB1) of RNA polymerase II also contains an essential C-terminal domain (CTD). RNA polymerases I and III contain the same two nonidentical a-like subunits, whereas RNA polymerase II contains two other nonidentical a-like subunits. All three polymerases share the same ra-like subunit and four other common subunits. In addition, each yeast polymerase contains three to seven unique smaller subunits.

(Figure 11-6). Each of the eukaryotic polymerases also contains an ra-like and two nonidentical a-like subunits. The extensive similarity in the structures of these core subunits in RNA polymerases from various sources indicates that this enzyme arose early in evolution and was largely conserved. This seems logical for an enzyme catalyzing a process so basic as copying RNA from DNA.

In addition to their core subunits related to the E. coli RNA polymerase subunits, all three yeast RNA polymerases contain four additional small subunits, common to them but not to the bacterial RNA polymerase. Finally, each eukary-otic polymerase has several enzyme-specific subunits that are not present in the other two polymerases. Gene-knockout experiments in yeast indicate that most of these subunits are essential for cell viability. Disruption of the few polymerase subunit genes that are not absolutely essential for viability nevertheless results in very poorly growing cells. Thus it seems likely that all the subunits are necessary for eukaryotic RNA polymerases to function normally.

The Largest Subunit in RNA Polymerase II Has an Essential Carboxyl-Terminal Repeat

The carboxyl end of the largest subunit of RNA polymerase II (RPB1) contains a stretch of seven amino acids that is nearly precisely repeated multiple times. Neither RNA poly-merase I nor III contains these repeating units. This hep-tapeptide repeat, with a consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser, is known as the carboxyl-terminal domain (CTD). Yeast RNA polymerase II contains 26 or more repeats, the mammalian enzyme has 52 repeats, and an intermediate number of repeats occur in RNA polymerase II from nearly all other eukaryotes. The CTD is critical for viability, and at least 10 copies of the repeat must be present for yeast to survive.

▲ EXPERIMENTAL FIGURE 11-7 Antibody staining demonstrates that the carboxyl-terminal domain (CTD) of RNA polymerase II is phosphorylated during in vivo transcription. Salivary gland polytene chromosomes were prepared from Drosophila larvae just before molting. The preparation was treated with a rabbit antibody specific for phosphorylated CTD and with a goat antibody specific for unphosphorylated CTD. The preparation then was stained with fluorescein-labeled anti-goat antibody (green) and rhodaminelabeled anti-rabbit antibody (red). Thus polymerase molecules with an unphosphorylated CTD stain green, and those with a phosphorylated CTD stain red. The molting hormone ecdysone induces very high rates of transcription in the puffed regions labeled 74EF and 75B; note that only phosphorylated CTD is present in these regions. Smaller puffed regions transcribed at high rates also are visible. Nonpuffed sites that stain red (up arrow) or green (horizontal arrow) also are indicated, as is a site staining both red and green, producing a yellow color (down arrow). [From J. R. Weeks et al., 1993, Genes & Dev. 7:2329; courtesy of J. R. Weeks and A. L. Greenleaf.]

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