Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II.

Abstract

The carboxyl-terminal domain (CTD) of the mouse RNA polymerase II largest subunit consists of 52 repeats of a seven-amino-acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A genetic approach was used to determine whether the CTD plays an essential role in RNA polymerase function. Deletion, insertion, and substitution mutations were created in the repetitive region of an aL-amanitin-resistant largest-subunit gene. The effects of these mutations on RNA polymerase II activity were assayed by measuring the ability of mutant genes to confer aL-amanitin resistance after transfection of susceptible rodent cells. Mutations that resulted in CTDs containing between 36 and 78 repeats had no effect on the transfer of a-amanitin resistance, whereas mutations with 25 or fewer repeats were inactive in this assay. Mutations that contained 29, 31, or 32 repeats had an intermediate effect; the number of a-amanitin-resistant colonies was lower and the colonies obtained were smaller, indicating that the mutant RNA polymerase II was defective. In addition, not all of the heptameric repeats were functionally equivalent in that repeats that diverged in up to three amino acids from the consensus sequence could not substitute for the conserved heptamer repeats. We concluded that the CTD is essential for RNA polymerase II activity, since substantial mutations in this region result in loss of function.The genes for several eucaryotic RNA polymerase subunits have been cloned and sequenced (1,2,6,8,10,12,20). These studies have revealed that the two largest subunits (IHo and IIc) of RNA polymerase TI are related to the ,B' and P subunits, respectively, of Escherichia coli RNA polymerase (2,8,11,12,27). Genetic and biochemical studies of the procaryotic transcription apparatus have shown that the ,B and P' subunits, together with two a subunits, make up the catalytic core of RNA polymerase (29, 30). Conservation of P-and P'-homologous sequences among eucaryotic subunits indicates that the basic catalytic core functions provided by these subunits have been maintained through evolution.The largest subunit of eucaryotic RNA polymerase II (RPII215) contains an additional domain for which no procaryotic counterpart exists. This domain consists of a repeated amino acid block with the consensus sequence TyrSer-Pro-Thr-Ser-Pro-Ser (see Fig. 1D). This sequence is repeated 26 times in Saccharomyces cerevisiae (2, 18a), about 40 times in Drosophila melanogaster (15), and 52 times in the mouse (11) and Chinese hamster (2a). The S. cerevisiae repetitive domain is made up almost entirely of consensus repeats (2), while the Drosophila domain shows more variability from the consensus sequence (15). In the mammalian repetitive domain, some repeats adhere strongly to the consensus sequence, whereas other repeats diverge considerably (2a, 11).The evolutionary conservation of the RNA polymerase II carboxyl-terminal domain (CTD) among eucaryotic species and the fact that no similar domain is found in the equivalent subunits of RNA polymerases I...

Evolution of the RNA polymerase II C-terminal domain

“…Our hypothesis of extreme stabilizing selection in members of the CTD-clade is in agreement with evidence from in vivo genetic investigations that show the CTD to be essential for viability in both complex multicellular metazoans, as well as more developmentally simple unicellular fungi (40)(41)(42). Thus, if heptapeptide repeats were present in the ancestors of most extant eukaryotic taxa, they must not have evolved under the same strict functional constraints in lineages outside the CTDclade.…”

Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases

“…The dierences in phosphorylation of the recombinant CTD substrate are less dramatic than those with the lysine-substituted peptide, presumably due to the presence of non-lysine-substituted repeats, but are still signi®cant. It would be interesting to examine the eects of removing or adding such non-consensus repeats to the CTD in an in vivo assay (Bartolomei et al, 1988). In addition, analysing the eects of CTD phosphorylation on Plasmodium berghei RNA polymerase II, which has a high number of lysine substitutions at the same position (Giesecke et al, 1991), may provide insight into the roles of cyclin C/CDK8 and cyclin H/CDK7/p36.…”

“…It consists of multiple, near-perfect tandem repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser, and a minimum number of these CTD heptapeptide repeats is required for RNA polymerase II activity in vivo and for cell viability (Allison et al, 1988;Bartolomei et al, 1988;Nonet et al, 1987;Zehring et al, 1988). RNA polymerase II becomes heavily phosphorylated during the course of transcription, and this phosphorylation mostly occurs on the CTD (Cadena and Dahmus, 1987;Dahmus, 1981).…”

Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing.

“…The number of repeats ranges from 26 in yeast, to 42 in Drosophila, to 52 in mammals. The CTD has been shown to carry out essential in vivo roles in yeast (Nonet et al 1987), Drosophila (Zehring et al 1988), and mammalian cells (Bartolomei et al 1988), but precisely what those roles are is not well understood. Suggested roles for the CTD include interacting with transcription initiation factors, serving as a molecular "cowcatcher" to facilitate movement of polymerase on chromatin templates, providing a link between transcription and RNA processing, and localizing polymerase to specific nuclear compartments (Corden 1990 (Allison et al 1988;Scafe et al 1990;Peterson et al 1991), suggesting that the CTD may be involved in receiving regulatory signals at certain promoters.…”