TFIIF (RAP30/74) is a general initiation factor that also increases the rate of elongation by RNA polymerase II. A two-hybrid screen for RAP74-interacting proteins produced cDNAs encoding FCP1a, a novel, ubiquitously expressed human protein that interacts with the carboxyl-terminal evolutionarily conserved domain of RAP74. Related cDNAs encoding FCP1b lack a carboxylterminal RAP74-binding domain of FCP1a. FCP1 is an essential subunit of a RAP74-stimulated phosphatase that processively dephosphorylates the carboxyl-terminal domain of the largest RNA polymerase II subunit. FCP1 is also a stoichiometric component of a human RNA polymerase II holoenzyme complex. Initiation of transcription by RNA polymerase (RNAP)1 II involves the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (reviewed in Ref. 1). Beginning with TFIID, whose TATA box-binding protein subunit recognizes the TATA box present in many promoters, these factors can assemble in an ordered pathway in vitro onto a promoter (2, 3), resulting in the formation of a preinitiation complex containing more than 40 polypeptides. Subsequently, however, yeast and mammalian RNAP II holoenzymes that contain several or all of the general transcription factors and other polypeptides were discovered (4 -9). There is evidence that transcription by RNAP II in Saccharomyces cerevisiae generally depends on such a holoenzyme (10) and that recruitment of yeast holoenzyme to a promoter would lead to a high rate of transcription (11).During or shortly after initiation by RNAP II, the carboxylterminal domain (CTD) of its largest subunit becomes heavily phosphorylated and remains so during transcript elongation (12). The phosphorylated form of RNAP II is designated RNAP IIO, whereas the unphosphorylated form is designated RNAP IIA. One subunit of TFIIH is a protein kinase that can phosphorylate the CTD (13). Phosphorylation of the CTD by PTEFb, a different Drosophila CTD kinase, has been shown to enhance the processivity of chain elongation by RNAP II in vitro (14). Concomitant with or following the termination of transcription, the CTD must be dephosphorylated by a protein phosphatase, since RNAP IIO cannot assemble directly into a preinitiation complex on either the adenovirus-2 major late or murine dihydrofolate reductase promoter in vitro (15-17). Accordingly, CTD phosphatase may function as a global regulator of gene expression by controlling the pool of RNAP IIA available for initiation. A phosphatase whose activity is stimulated by RAP74 and dephosphorylates the CTD in a processive manner has been purified from HeLa cell extracts (18,19).Certain activator proteins increase the efficiency of RNA chain elongation downstream from the promoter. For example, RNAP II pauses with an unphosphorylated CTD about 25-40 nucleotides downstream from the initiation site of Drosophila hsp70 genes and is stimulated by heat shock and the heat shock factor to become phosphorylated and leave these pause sites (Ref. 20 and references therein). Increasing evidence suppo...

The phosphorylation state of the carboxyl-terminal domain (CTD) of RNA polymerase (RNAP) II is directly linked to the phase of transcription being carried out by the polymerase. Enzymes that affect CTD phosphorylation can thus play a major role in the regulation of transcription. A previously characterized HeLa CTD phosphatase has been shown to processively dephosphorylate RNAP II and to be stimulated by the 74-kDa subunit of TFIIF. This phosphatase is shown to be comprised of a single 150-kDa subunit by the reconstitution of catalytic activity from a SDS-polyacrylamide gel electrophoresis purified protein. This subunit has been previously cloned and shown to interact with the HIV Tat protein. To determine whether this interaction has functional consequences, the effect of Tat on CTD phosphatase was investigated. Full-length Tat-1 protein (Tat 86R) strongly inhibits the activity of CTD phosphatase. Point mutations in the activation domain of Tat 86R, which reduce the ability of Tat to transactivate in vivo, diminish its ability to inhibit CTD phosphatase. Furthermore, a deletion mutant missing most of the activation domain is unable to inhibit CTD phosphatase activity. The ability of Tat to transactivate in vitro also correlates with the strength of inhibition of CTD phosphatase. These results are consistent with the hypothesis that Tat-dependent suppression of CTD phosphatase is part of the transactivation function of Tat.The largest subunit of RNA polymerase (RNAP) 1 II from all eukaryotes contains at its carboxyl terminus varying numbers of repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-ProSer. Each cycle of transcription involves the reversible phosphorylation of this carboxyl-terminal domain (CTD) (1). RNAP II with an unphosphorylated CTD is designated RNAP IIA, and polymerase with a highly phosphorylated CTD is designated RNAP IIO. The activity of many key regulatory factors appears to be mediated by the CTD although its role in transcription is not clearly defined. An understanding of transcriptional regulation is dependent on elucidating the function of the CTD and the mechanisms and regulation of the enzymes that modify it.Because RNAPs IIA and IIO have distinct roles in the transcription cycle, the activities of enzymes that phosphorylate and dephosphorylate the CTD must be tightly regulated. RNAP IIA is efficiently recruited to promoters during the assembly of a preinitiation complex (2-6) whereas RNAP IIO is associated with the elongation complex (7,8). Therefore, at some point subsequent to the binding of RNAP II to the promoter and likely before RNAP II has cleared the promoter, the CTD is phosphorylated by CTD kinase(s) (2). After completion of a nascent transcript, RNAP IIA must be regenerated by CTD phosphatase. An enzyme capable of selectively dephosphorylating the CTD has been described (9 -12).CTD phosphatases and kinases can be inhibitory or stimulatory to transcription depending on the phase of the transcription cycle during which they act. According to present models, the phosphor...

Reversible phosphorylation of the C-terminal domain (CTD) of the largest RNA polymerase II (RNAP II) subunit plays a key role in gene expression. Stresses such as heat shock result in marked changes in CTD phosphorylation as well as in major alterations in gene expression. CTD kinases and CTD phosphatase(s) contribute in mediating differential CTD phosphory-lation. We now report that heat shock of HeLa cells at temperatures as mild as 41 degreesC results in a decrease in CTD phosphatase activity in cell extracts. The obser-vation that this CTD phosphatase interacts with the RAP74 subunit of the general transcription factor TFIIF suggests that it corresponds to the previously charac-terized major CTD phosphatase. This conclusion is also supported by the finding that the distribution of the 150 kDa subunit of CTD phosphatase in cells is altered by heat shock. Although CTD phosphatase is found predominantly in low salt extracts in unstressed cells, immunofluorescence microscopy indicates that its intracellular localization is nuclear. The decrease in CTD phosphatase activity correlates with a decrease in amount of 150 kDa phosphatase subunit in the extracts. During heat shock, CTD phosphatase switches to an insoluble form which remains aggregated to the nuclear matrix fraction. In contrast, heat shock did not result in a redistribution of RAP74, indicating that not all nuclear proteins aggregate under these conditions. Accordingly, the heat-inactivation of both the CTD phosphatase and the TFIIH-associated CTD kinase might contribute to the selective synthesis of heat-shock mRNAs.

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and Summary.-Chromosomal RNA, which is associated with chromosomal proteins in the chromosomes of higher organisms, possesses the ability to hybridize to homologous native DNA. The proportion of native DNA thus hybridized is similar to the proportion of denatured DNA which hybridizes with chromosomal RNA, and both are similar to the proportions of chromosomal RNA and DNA in native chromatin.We have found that chromosomal RNA possesses the ability to hybridize to homologous native DNA. Chromosomal RNA is a particular class of RNA distinguished by short chain length (40-60 nucleotides in length in different organisms),1' 2 high (8-10 mole %) content of dihydroprimidine (dihydrouridylic acid in pea, cow, and chick, dihydroribothymidylic acid in rat),3 and by its association with DNA in the chromosomes of higher organisms. In such chromosomes, chromosomal RNA is on the one hand bound to DNA in RNase-resistant form,4 and on the other is bound covalently to chromosomal protein. 3 We have previously shown that chromosomal RNA is sequence-heterogeneous and hybridizes to the extent of 3-5 per cent to homologous denatured DNA, the exact percentage depending upon the organism involved.2' 6 We show below that chromosomal RNA hybridizes to this same extent with homologous native DNA.Materials and Methods.-Preparation of chromatin: All of the experiments reported below were done with chromosomal RNA of rat Novikoff ascites tumor. The chromatin was prepared from tumor cells by the method of Dahmus and McConnell.2 The ascites cells were removed from the rat on the fifth to sixth day after infection with the tumor and were immediately washed by pelleting for 6 min at 700 g in TNKM buffer (0.05 M tris, pH 6.7, 0.13 M NaCl, 0.025 M KCl, 0.0025 M MgCl2), followed by a single pelleting from 4X-diluted TNKM buffer. The cells were then lysed in deionized water and the nuclei pelleted at 1500 g for 15 min. The nuclear pellet was homogenized by hand in a Teflon homogenizer and in 0.01 M tris, pH 8.0. The chromatin was pelleted and washed by repelleting four times from the same buffer. The resulting crude chromatin was then purified by layering it on 1.7 M sucrose and pelleting it for 2 hr at 22,000 rpm in the Spinco SW25 rotor.Preparation of chromosomal RNA: The sucrose density gradient-purified chromatin, dialyzed against 0.01 M Tris to free it of sucrose, was dissolved in 4 M CsCl and centrifuged for 18 hr at 35,000 rpm in the Spinco no. 40 rotor. Under these conditions, DNA and messenger RNA pellet, while chromosomal proteins, together with chromosomal RNA, float as a skin or pellicle.6 The chromosomal protein-RNA skin was next washed free of CsCl by pelleting it from 70% EtOH, suspended in 0.01 All tris pH 8.0, and protein-digested at 370C with preincubated (90 min, 370C) pronase, final concentration 1 mg/ml, for 4 hr at 370C. Any undigested and aggregated protein was centrifuged off; the RNA phenol-extracted in the cold and then precipitated from 0.2 N KAc by addition of 2 vol of EtOH. The RNA, redissolved in 7 Al urea co...