Heat shock of HeLa cells inactivates a nuclear protein phosphatase specific for dephosphorylation of the C-terminal domain of RNA polymerase II.

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.

Regulation of carboxyl-terminal domain phosphatase by HIV-1 tat protein.

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.

FCP1, the RAP74-interacting subunit of a human protein phosphatase that dephosphorylates the carboxyl-terminal domain of RNA polymerase IIO.

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 carboxyl-terminal 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.

Similarities in structure and function of calf thymus and Drosophila casein kinase II.

Both calf and Drosophila contain a type II casein kinase with similar molecular structure and catalytic activity. Purified calf thymus casein kinase II is composed of three subunits of Mr = 44,000 (alpha), 40,000 (alpha'), and 26,000 (beta) (Dahmus, M.E. (1981) J. Biol. Chem. 256, 3319-3325), whereas the Drosophila enzyme is composed of two subunits of Mr = 36,700 (alpha) and 28,200 (beta) (Glover, C. V. C., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 3258-3265). The native form of the enzyme is an alpha 2 beta 2 tetramer. Polyclonal antibodies prepared against each enzyme react with both the alpha and beta subunits of the homologous enzyme and cross-react with both subunits of the heterologous enzyme. Reaction of polyclonal antibodies with proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis establishes that no significant difference in subunit molecular weight exists between the purified enzymes and the enzyme present in initial cell extracts. Each antibody effectively inhibits the in vitro activity of the homologous enzyme and causes a slight inhibition in the activity of the heterologous enzyme. Peptide maps derived from purified subunits indicate that the alpha and beta subunits are unique and that there is extensive primary sequence homology between the corresponding subunits of the calf and Drosophila enzyme. Casein kinase II from both sources phosphorylates the same subunits of calf thymus RNA polymerase II and an identical set of proteins in a complex mixture of acid-soluble proteins from Drosophila tissue culture cells. The striking similarity in molecular structure and catalytic activity between the calf and Drosophila enzyme suggests that casein kinase II has been highly conserved in evolution.

Hybridization of chromosomal RNA to native DNA.

Chromosomal RNA, which is associated with chromosomal proteins in the chromosomes of higher organisms, possess 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.