To investigate functional differences between RNA polymerases IIA and II0 (Pol IIA and Pol II0), with hypoand hyperphosphorylated carboxy-terminal repeat domains (CTDs), respectively, we have visualized the in vivo distributions of the differentially phosphorylated forms of Pol II on Drosophila polytene chromosomes.Using phosphorylation state-sensitive antibodies and immunofluorescence microscopy with digital imaging, we find Pol IIA and Pol II0 arrayed in markedly different, locus-and condition-specific patterns. Major ecdysone-induced puffs, for example, stain exclusively for Pol II0, indicating that hyperphosphorylated Pol II is the transcriptionally active form of the enzyme on these genes. In striking contrast, induced heat shock puffs stain strongly for both Pol IIA and Pol II0, suggesting that heat shock genes are transcribed by a mixture of hypo-and hyperphosphorylated forms of Pol II. At the insertion sites of a transposon carrying a hybrid hsp70-lacZ transgene, we observe only Pol IIA before heat shock induction, consistent with the idea that Pol II arrested on the hsp70 gene is form IIA. After a 90-sec heat shock, we detect heat shock factor (HSF) at the transposon insertion sites; and after a 5-min shock its spatial distribution on the induced transgene puffs is clearly resolved from that of Pol II. Finally, using antibodies to hnRNP proteins and splicing components, we have discerned an apparent overall correlation between the presence and processing of nascent transcripts and the presence of Pol II0.[Key Words: RNA polymerase IIA/II0; CTD phosphorylation; transcription factors; in situ localization; immunofluorescence microscopy; digital imaging] Received July 28, 1993; revised version accepted September 29, 1993.The carboxy-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is an unusual entity composed of multiple repeats of the 7-amino-acid consensus sequence YSPTSPS (for review, see Corden 1990;Young 1991). 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. In vivo and in vitro experiments suggest that these responses might be mediated through the TATA-binding protein (TBP) component of TFIID (Koleske et al. 1992;Usheva et al. 1992;Thompson et al. 1993). On the other hand, the precise nature of the CTD involvement in initiation is not known, ...
A chemical genetics approach identified a cellular target of several proapoptotic farnesyl transferase inhibitors (FTIs). Treatment with these FTIs caused p53-independent apoptosis in Caenorhabditis elegans, which was mimicked by knockdown of endosomal trafficking proteins, including Rab5, Rab7, the HOPS complex, and notably the enzyme Rab geranylgeranyl transferase (RabGGT). These FTIs were found to inhibit mammalian RabGGT with potencies that correlated with their proapoptotic activity. Knockdown of RabGGT induced apoptosis in mammalian cancer cell lines, and both RabGGT subunits were overexpressed in several tumor tissues. These findings validate RabGGT, and by extension endosomal function, as a therapeutically relevant target for modulation of apoptosis, and enhance our understanding of the mechanism of action of FTIs.
Saccharomyces cerevisiae CTDK-I is a protein kinase complex that specifically and efficiently hyperphosphorylates the carboxyl-terminal repeat domain (CTD) of RNA polymerase II and is composed of three subunits of 58, 38, and 32 kDa. The kinase is essential in vivo for normal phosphorylation of the CTD and for normal growth and differentiation. We have now cloned the genes for the two smaller kinase subunits, CTK2 and CTK3, and found that they form a unique, divergent cyclin-cyclin-dependent kinase complex with the previously characterized largest subunit protein CTK1, a cyclin-dependent kinase homolog. The CTK2 gene encodes a cyclin-related protein with limited homology to cyclin C, while CTK3 shows no similarity to other known proteins. Copurification of the three gene products with each other and CTDK-I activity by means of conventional chromatography and antibody affinity columns has verified their participation in the complex in vitro. In addition, null mutations of each of the genes and all combinations thereof conferred very similar growth-impaired, cold-sensitive phenotypes, consistent with their involvement in the same function in vivo. These characterizations and the availability of all of the genes encoding CTDK-I and reagents derivable from them will facilitate investigations into CTD phosphorylation and its functional consequences both in vivo and in vitro.
The unique C-terminal repeat domain (CTD) of the largest subunit (lla) of eukaryotic RNA polymerase II consists of multiple repeats of the heptapeptide consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats ranges from 26 in yeast to 42 in Drosophila to 52 in mouse. The CTD is essential in vivo, but its structure and function are not yet understood. The CTD can be phosphorylated at multiple serine and threonine residues, generating a form of the largest subunit (11o) with markedly reduced mobility in NaDodSO4/polyacrylamide gels. To investigate this extensive phosphorylation, which presumably modulates functional properties of RNA polymerase II, we began efforts to purify a specific CTD kinase. Using CTD-containing fusion proteins as substrates, we have purified a CTD kinase from the yeast Saccharomyces cerevisiae. The enzyme extensively phosphorylates the CTD portion of both the fusion proteins and intact subunit IIa, producing products with reduced electrophoretic mobilities. The properties of the CTD kinase suggest that it is distinct from previously described protein kinases. Analogous activities were also detected in Drosophila and HeLa cell extracts.Eukaryotic RNA polymerase II is a complex enzyme consisting of [8][9][10][11][12] (2,3,9): (i) interacting with certain transcription factors during initiation; (ii) anchoring RNA polymerase II to a structure within the nucleus; (iii) destabilizing of histone-DNA interactions during elongation (phosphorylated form Ho). The CTD is essential for cell viability (4,(10)(11)(12), but its precise function has not been elucidated. RNA polymerase II completely lacking the CTD is capable of initiating transcription at promoter sites in at least one factor-dependent in vitro system (4). On the other hand, antibody inhibition (13,14) and photoaffinity labeling (15, 16) experiments support the idea that the CTD, probably in its phosphorylated form, plays an important role in specific transcription.As a step toward understanding the roles of CTD phosphorylation in vivo, we began efforts to purify protein kinases that phosphorylate this domain. We purified CTD-containing fusion proteins from Escherichia coli and used them as substrates to detect an activity that extensively phosphorylates the CTD. Here we report the purification and preliminary characterization of this CTD kinase from the yeast Saccharomyces cerevisiae. METHODSConstruction and Purification of CTD Fusion Proteins. For the yeast CTD fusion protein construction, a 1.05-kilobase (kb) Bgl II-HindIII fragment from RP021 (2) that encodes the entire CTD and 48 upstream amino acids was isolated from plasmid pJH107 (from C. J. Ingles, University of Toronto) and inserted into the BamHh-HindhIh site in pUR290 (17). For the Drosophila fusion protein the 3.6-kb EcoRI fragment from RpII215 encoding the CTD (18) was subcloned into pBR325, and the resulting 2.3-kb Sal 1-HindIIl fragment, containing 34 of the total 42 repeats (19), was inserted into the Sal I-HindIII site in pUR290. These recombinan...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.