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...

The interleukin-2 receptor alpha (IL-2R alpha) chain gene contains a sequence similar to the immunoglobulin (Ig) kappa (kappa) enhancer NF-kappa B binding site. This site, which is bound by the nuclear protein, NF-kappa B, is critical for Ig kappa gene expression. The major T cell nuclear factor that binds to the IL-2R alpha site in vitro appears indistinguishable from NF-kappa B. NF-kappa B binds to IL-2R alpha and kappa sequences with similar affinities; however, only the kappa site potently activates transcription from heterologous promoters. Thus, high-affinity NF-kappa B binding in vitro cannot be equated with transcriptional activation in vivo. Mutation of the NF-kappa B binding site in the context of an IL-2 R alpha promoter construct markedly diminished promoter activity in human T cell lymphotropic virus type I (HTLV-I)-transformed MT-2 cells but not in phorbol myristate acetate-stimulated Jurkat T cells.

High-affmity receptors for interleukin 2 (IL-2) are expressed on T cells following activation. These receptors are composed of both a and (3 chains. Expression of a chains and, therefore, expression of high-affinity receptors are critically regulated at the level of transcription initiation. We have further dissected the regulatory elements involved in controlling transcription of the IL-2 receptor a-chain (IL-2Ra) gene. The IL-2Ra promoter contains a KB site and binding sites for additional nuclear factors within a 50-base-pair region (positions -290 to -240 relative to the major transcription start site). These include one upstream of the KB site and one similar to the c-fos serum response element (SRE), which is downstream of the KB site. Mutation ofthe KB site decreases IL-2Ra promoter activity in MT-2 cells (a T-cell line that has been transformed with human T-cell lymphotropic virus type I), but not in Jurkat cells (a T-cell leukemia line) that have been activated by phorbol 12-myristate 13-acetate (PMA). In contrast, mutation of a region upstream of the KB site decreases activity in PMA-induced Jurkat cells but increases activity in MT-2 cells. Mutation of the SRE-llke site decreases activity in both cell types but the effect in PMA-induced Jurkat is more pronounced. Thus, these distinct cis-acting elements play different physiological roles in IL-2Ra gene activation in MT-2 cells and PMA-induced Jurkat T cells. These studies provide direct evidence for a functionally significant SRE-like sequence in a gene other than c-fos and the actin genes and identify other elements that are critical for IL-2Ra gene expression.

We have delineated a positive regulatory element in the interleukin-2 receptor a-chain gene between positions -299 and -243 that can potently activate a heterologous (herpesvirus thymidine kinase [tk]) promoter in phorbol myristate acetate (PMA)-induced Jurkat T cells and is functional when cloned in either orientation. This enhancerlike element contains a site (-268/-257) that can bind NF-KB; however, unlike the immunoglobulin K gene KB enhancer element, the IL-2Ra KB-like site alone can only weakly activate a heterologous promoter. Adjacent 5' and 3' sequences also weakly activate the tk-CAT vector, but constructs combining the IL-2Ra KB-like site plus adjacent 5' and 3' sequences potently activate gene expression. This combination of regions is essential for potent PMA-induced transcription from the tk promoter. Experiments using constructs in which IL-2Ra upstream sequences are sequentially deleted suggested that there is a region 5' of position -299 which can suppress IL-2Ra promoter and/or enhancer activity. Thus, it is possible that both positive and negative elements may be important in the regulation of IL-2Ra gene transcription.