Molecular cloning and characterization of rpbA encoding RNA polymerase II largest subunit from a filamentous fungus, Aspergillus oryzae

We have cloned rpbA encoding the RNA polymerase II largest subunit (polIIL) from a filamentous fungus, Aspergillus oryzae. The rpbA product included eight highly conserved regions and the carboxyl-terminal domain (CTD). A. oryzae polIIL CTD with 184 amino acids was composed of 25 CTD consensus repeats, which was a similar number to those of lower eukaryotes. The amino acids in each repeat of A. oryzae polIIL, however, conformed less to the CTD consensus than those of polIILs from other lower eukaryotes.     

Functional unit of the RNA polymerase II C-terminal domain lies within heptapeptide pairs

Unlike all other RNA polymerases, the largest subunit (RPB1) of eukaryotic DNA-dependent RNA polymerase II (RNAP II) has a C-terminal domain (CTD) comprising tandemly repeated heptapeptides with the consensus sequence Y-S-P-T-S-P-S. The tandem structure, heptad consensus, and most key functions of the CTD are conserved between yeast and mammals. In fact, all metazoans, fungi, and green plants examined to date, as well as the nearest protistan relatives of these multicellular groups, contain a tandemly repeated CTD. In contrast, the RNAP II largest subunits from many other eukaryotic organisms have a highly degenerate C terminus or show no semblance of the CTD whatsoever. The reasons for intense stabilizing selection on CTD structure in certain eukaryotes, and its apparent absence in others, are unknown. Here we demonstrate, through in vivo genetic complementation, that the essential functional unit of the yeast CTD is contained within pairs of heptapeptides. Insertion of a single alanine residue between diheptads has little phenotypic effect, while increasing the distance between diheptads produces a mostly quantitative effect on yeast cell growth. We further explore structural constraints on the CTD within an evolutionary context and propose selective mechanisms that could maintain a global tandem structure across hundreds of millions of years of eukaryotic evolution.