RNA Polymerases IIA Research Articles

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

To investigate functional differences between RNA polymerases IIA and IIO (Pol IIA and Pol IIO), with hypo- and 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 IIO arrayed in markedly different, locus- and condition-specific patterns. Major ecdysone-induced puffs, for example, stain exclusively for Pol IIO, 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 IIO, 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 IIO.

RNA polymerases IIA and IIO have distinct roles during transcription from the TATA-less murine dihydrofolate reductase promoter.

The largest subunit of RNA polymerase II (RNAP II) contains a remarkable region of tandem heptapeptide repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser at its carboxyl terminus. This COOH-terminal domain (CTD) is unphosphorylated in RNAP IIA, extensively phosphorylated in RNAP IIO, and absent in RNAP IIB. The reversible phosphorylation of the CTD has been proposed to be integral to each cycle of transcription from the adenovirus-2 major late promoter. The adenovirus-2 major late promoter, however, may not be a good paradigm for the study of CTD function because in vitro transcription from this promoter is not dependent on the CTD. Previous studies suggest that transcription from the murine dihydrofolate reductase (DHFR) promoter requires the CTD. In an effort to investigate the role of the CTD and its phosphorylation, a RNAP II-dependent reconstituted transcription system specific for the DHFR promoter was established. In this reconstituted system, RNAP IIA, but not RNAP IIB, can transcribe from the DHFR promoter. Furthermore, RNAP IIB does not compete with RNAP IIA for preinitiation complex assembly. These results suggest that the CTD plays a critical role in the recruitment of RNAP II to the DHFR promoter. The analysis of preinitiation complexes assembled on the DHFR promoter indicates that RNAP IIA readily assembles into functional preinitiation complexes in contrast to the inefficient assembly of RNAP IIO. However, transcript elongation is catalyzed by RNAP IIO as demonstrated by the photoactivated cross-linking of nascent DHFR transcripts to subunit IIo. These results indicate that transcription from the DHFR promoter involves the reversible phosphorylation of the CTD and support the idea that RNAPs IIA and IIO have essential but distinct functions.

The interaction of RNA polymerase II with the adenovirus-2 major late promoter is precluded by phosphorylation of the C-terminal domain of subunit IIa.

Mammalian RNA polymerase II contains at the C terminus of its largest subunit an unusual domain consisting of 52 tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The phosphorylation of this domain is thought to play an important role in the transition of RNA polymerase II from a preinitiation complex to an elongating complex. The unphosphorylated form of RNA polymerase II is designated IIA, whereas the phosphorylated form is designated IIO. In an effort to determine the consequence of C-terminal domain phosphorylation on complex formation, 32P-labeled RNA polymerases IIA and IIO were prepared and examined for their ability to form a stable preinitiation complex on the adenovirus-2 major late promoter in the presence of a reconstituted HeLa cell transcription extract. Preinitiation complexes were formed in the absence of ATP and purified from free RNA polymerase II by chromatography on Sepharose CL-4B. The state of phosphorylation of the largest subunit was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the transcriptional activity was determined by assaying specific transcript formation upon the addition of nucleotides and a competing DNA template. RNA polymerase IIA was recovered in transcriptionally active complexes in reactions in which the input enzyme was RNA polymerase IIA. In reactions with RNA polymerase IIO as the input enzyme, no IIO was recovered in excluded fractions that normally contain preinitiation complex. In reactions with equimolar amounts of RNA polymerases IIO and IIA, purified preinitiation complexes contained almost exclusively RNA polymerase HA. These results support the idea that RNA polymerase II containing an unphosphorylated C-terminal domain preferentially associates with the adenovirus-2 major late promoter. The state of phosphorylation of the C-terminal domain can, therefore, directly influence preinitiation complex formation. We also report here the presence of an activity in HeLa cell extracts that catalyzes dephosphorylation of the C-terminal domain, thereby converting RNA polymerase IIO to IIA. This C-terminal domain phosphatase is specific in that it does not catalyze the dephosphorylation of a serine residue phosphorylated by casein kinase II. The presence of a C-terminal domain phosphatase in in vitro transcription reactions containing RNA polymerase IIO results in the formation of RNA polymerase IIA. This RNA polymerase IIA associates preferentially with preinitiation complexes.

The Major Late Promoter of Adenovirus-2 Is Accurately Transcribed by RNA Polymerases IIO, IIA, and IIB

Subunit IIa of mammalian RNA polymerase II contains at its C terminus 52 tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain is unmodified in RNA polymerase IIA, extensively phosphorylated in RNA polymerase IIO, and absent from RNA polymerase IIB. In an effort to define the role of the C-terminal domain, we have measured the transcriptional activity of purified RNA polymerases IIO, IIA, and IIB. The ability of each polymerase subspecies to transcribe the major late promoter of adenovirus-2 was examined in a polymerase-dependent transcription system reconstituted from partially purified transcription factors. RNA polymerases IIO, IIA, and IIB are all capable of initiating specific transcripts from this promoter. The transcriptional activity was determined as a function of the concentration of RNA polymerase II, template DNA, and each of the essential general transcription factors. The transcriptional activities of RNA polymerases IIA and IIB were comparable and consistently greater than that of RNA polymerase IIO when assayed under the conditions described here. The kinetics of transcript formation is similar except that RNA polymerase IIO has a more pronounced lag. These results show that the C-terminal domain of subunit IIa is not essential for the accurate initiation of transcripts from the major late promoter of adenovirus-2 and that the effect of the C-terminal domain is not likely mediated by the general transcription factors required for the expression of class II genes.

Purification of RNA polymerase IIO from calf thymus.

Three subspecies of RNA polymerase II, designated IIO, IIA, and IIB, have been described in calf thymus and shown to differ in the apparent molecular weight of their largest subunits, designated IIo, IIa, and IIb, respectively. The objective of this study was to develop a procedure for the purification of RNA polymerase IIO. This form of the enzyme predominates in vivo and is responsible for the transcription of most cellular genes. RNA polymerase II is solubilized from isolated calf thymus nuclei in the presence of high concentrations of chelators, precipitated with polyethyleneimine, extracted with salt, and precipitated with (NH4)2SO4. The solubilized enzyme is resolved from factors that destabilize RNA polymerase IIO by chromatography on heparin-Sepharose CL-4B and DE52. RNA polymerase IIO is then partially resolved from RNA polymerases IIA and IIB by chromatography on DEAE-5PW and further purified by chromatography on Phenyl-Superose and Mono Q. RNA polymerase IIO was purified 1000-fold from the polyethyleneimine eluate resulting in about 130 micrograms of RNA polymerase IIO from 300 g of calf thymus. The specific activity of RNA polymerase IIO, in nonselective assays using calf thymus DNA as template, is 440 units/mg and not significantly different from that of RNA polymerases IIA and IIB. The similar transcriptional activities in nonselective assays suggest that the C-terminal domain of the largest RNA polymerase II subunit does not play a major role in the elongation phase of the reaction when deproteinized DNA serves as template. The small subunits of RNA polymerase IIO are indistinguishable from those of RNA polymerases IIA and IIB.

Immunochemical analysis of mammalian RNA polymerase II subspecies. Stability and relative in vivo concentration.

Three subspecies of RNA polymerase II, designated IIO, IIA, and IIB, have been described in a variety of eukaryotic cells and shown to differ in the molecular weight of their largest subunit, designated IIo, IIa, and IIb, respectively. The objectives of this study were to establish the in vivo molecular structure of RNA polymerase II in mammalian cells and to examine conditions that influence the stability of RNA polymerase II subspecies. Subunit affinity-purified antibodies were used to determine the relative concentration of subunits IIo, IIa, and IIb in crude extracts of calf thymus tissue, cultured bovine kidney cells, and HeLa cells. HeLa cells contain exclusively RNA polymerase IIO whereas both cultured bovine kidney cells and calf thymus tissue contain RNA polymerases IIO and IIA. RNA polymerase IIB was not detected at significant levels in any of the cell extracts examined. Cell extracts were aged at either 4 degrees or 37 degrees C and the stability of RNA polymerases IIO and IIA determined by protein blotting. In the presence of buffer normally used for RNA polymerase purification, subunit IIo disappears from calf thymus extracts within 24 h at 4 degrees C or within 5 min at 37 degrees C. RNA polymerase IIO is partially stabilized by the inclusion of protease inhibitors and further stabilized by the presence of relatively high concentration of EDTA and EGTA. The prior fractionation of nuclei does not have an appreciable effect on RNA polymerase II stability. An increase in the amount of reducing agent causes a dramatic reduction in the stability of subunit IIo. The following manuscript (Bartholomew, B., Dahmus, M. E., and Meares, C. F. (1986) J. Biol. Chem. 14226-14231) examines the transcriptional activity of RNA polymerases IIO and IIA in reactions dependent on the major late promoter of adenovirus-2. Photoaffinity labeling of subunits IIo and IIa, relative to their concentration in the transcription extract, indicates that the transcriptional activity of RNA polymerase IIO is greater than 10 times that of IIA.

Transcription of adenovirus-2 major late promoter inhibited by monoclonal antibody directed against RNA polymerases IIO and IIA.

Monoclonal antibody directed against RNA polymerases IIO and IIA completely inhibits specific transcription from the major late promoter of adenovirus-2. At comparable antibody concentrations, only a slight inhibition of polymerase III transcription of VA RNA I is observed. Inhibition of polymerase II transcription is specific for monoclonal IgM directed against RNA polymerase II. High concentrations of control IgM do not inhibit transcription. Specific transcription from the conalbumin and ovalbumin promoters is also inhibited by polymerase-specific monoclonal IgM but not control IgM. These results suggest that the in vitro transcription of the conalbumin, ovalbumin, and adenovirus-2 major late promoter is catalyzed by RNA polymerase(s) IIO and/or IIA but not RNA polymerase IIB. However, transcription from calf thymus DNA, known to contain nicks, is not inhibited by polymerase specific monoclonal antibody. Thus, the monoclonal antibody does not interfere with initiation or elongation of RNA polymerase from nonpromoter sites but does appear to selectively inhibit transcription from promoter sites.

Multiple forms of DNA-dependent RNA polymerases in Xenopus laevis. Rapid purification and structural and immunological properties.

DNA-dependent RNA polymerases I, II, and III (EC 2.7.7.6) were isolated from Xenopus laevis ovaries. The soluble enzymes were precipitated with polyethyleneimine and subjected to chromatography on heparin-Sepharose, DEAE-Sephadex, and phosphocellulose. RNA polymerase I was subjected to an additional chromatographic step on CM-Sephadex. The procedure required 40 h and produced purified RNA polymerase forms IA, IIA, and III in yields of 5 to 40%. The specific activities of RNA polymerases IIA and III (on native DNA) were comparable to those reported from other eukaryotic sources, whereas that of form IA was severalfold greater than the specific activities reported for other purified class I RNA polymerases. The complex subunit compositions of chromatographically purified RNA polymerases IA, IIA, and III were distinct when analyzed by polyacrylamide gradient gel electrophoresis under denaturing conditions, although all three classes contained polypeptides with Mr = 29,000, 23,000, and 19,000. Antibodies prepared against RNA polymerase III showed common antigenic determinants within the class I, II, and III enzymes. The sites responsible for the cross-reaction are located, at least in part, on the common 29,000-dalton polypeptide.

Monoclonal antibody specific for calf thymus RNA polymerases IIO and IIA.

Monoclonal antibody specific for calf thymus RNA polymerases IIO and IIA.

Plant DNA-dependent RNA polymerases: subunit structures and enzymatic properties of the class II enzymes from quiescent and proliferating tissues.

Class II DNA-dependent RNA polymerases were purified from soybean tissues of different physiological states: (1) from seed embryo tissue, representative of a quiescent, low metabolic state and (2) from auxin-treated hypocotyl tissue, representative of a highly proliferative and metabolically active state. Dodecyl sulfate, polyacrylamide gel electrophoresis indicates that RNA polymerase II from embryonic tissue consists largely (90-95%) of the form IIA enzyme, the largest subunit having a molecular weight of 215 000. RNA polymerase II from hypocotyl tissue is exclusively a form IIB enzyme, the largest subunit having a molecular weight of 180 000. Polypeptides common to RNA polymerases IIA and IIB have the following molecular weights: 138 000; 42 000; 27 000; 22 000; 19 000; 17 600; 17 000; 16 200; 16 100; and 14 000. Peptide mapping in the presence of dodecyl sulfate suggests that the 215 000 and 180 000 subunits possess similar peptide fragments. Plant embryo tissues do not contain protease activity capable of cleaving the 215 000 subunit to the 180 000 subunit, but proliferating plant tissues do contain such an activity. Mixing experiments indicate that appreciable amounts of RNA polymerase IIB are not being artifactually produced during protein purification.

Inhibition of nuclear DNA-dependent RNA polymerases from mouse ascites tumors and liver by glycerol.

Nuclear DNA-dependent RNA polymerases were isolated from Ehrlich ascites carcinoma, TA3 ascites adenocarcinoma, and mouse liver and tested for inhibition by glycerol. The results confirm the finding of Smith and Duerksen ((1975) Biochem. Biophys. Res. Commun. 67, 916-923) that glycerol may inhibit nuclear RNA polymerase II, but because different grades of glycerol inhibited mouse liver RNA polymerase IIa to different extents, it is suggested that an inhibitory contaminant is present. RNA polymerases IIa and IIb from the two tumors and mouse liver were proportionately inhibited by A.C.S. reagent-grade glycerol at concentrations above 10%. RNA polymerase Ia from liver and the TA3 tumor was not inhibited by any concentration of glycerol tested (2-32.3%), but RNA polymerase Ia from Ehrlich carcinoma was inhibited by glycerol concentrations above 16%.

The subunit structures of soluble and chromatin-bound RNA polymerase II from soybean

DNA-dependent RNA polymerase II is present in two forms, IIa and IIb, in germinating soybean. Form IIa is the dominant form of the enzyme in ungerminated embryos and appears to be a soluble enzyme. Form IIb increases in amount as germination progresses and is tightly bound to the chromatin template. The subunit structures of soybean RNA polymerases IIa and IIb are identical except for the molecular weights of their largest subunits which are 200,000 daltons and 170,000 daltons for IIa and IIb, respectively. The enzymes have seven common subunits: 142,000, 42,000, 26,000, 20,000, 16,000, 15,000, and 14,000 daltons.

Transcription of DNAs of known sequence after injection into eggs and oocytes of Xenopus laevis.

1. When the synthetic polynucleotide, poly[d(A-T) - d(A-T)] is injected into the eggs and oocytes of Xenopus laevis, a stimulation of RNA synthesis results. Analysis of extracted RNA by high-voltage electrophoresis, shows that this stimulation of RNA synthesis is due to the production of poly[r(A-U)] transcripts. The rate of poly[r(A-U)] synthesis is calculated to be at least ten-fold greater in eggs than in oocytes. The amount of poly[r(A-U)] detectable in injected eggs has reached a maximum by 1.5 h after injection; in oocytes, however, poly[r(A-U)] continues to accumulate between the third and 16th hour after injection. The transcripts range in length from less than 80 nucleotides up to over 2000 nucleotides long. The co-injection of poly[d(A-T)] - d(A-T)] and alpha-amanitin into oocytes, has shown that the synthesis of poly[r(A-U)] is approximately 90% inhibited at a concentration of alpha-amanitin which has no effect on the capacity of the oocyte to synthesize ribosomal and 4-S RNA; thus the nucleoplasmic RNA polymerases IIa and/or IIb, are implicated as playing a major role in poly[r(A-U)] synthesis in oocytes. 2. When poly(dG) - poly(d-C), poly(dA), poly(dA) - poly(dT) and poly[d(I-C) - d(I-C)] are individually injected into eggs only poly[d(I-C) - d(I-C)] is transcribed as efficiently as poly[d(A-T) - d(A-T)]. 3. When calf thymus native or denatured DNA, polyoma, T2, T4 and phiX DNAs are individually injected into eggs only the injection of calf thymus native DNA causes a detectable stimulation of RNA synthesis. 4. The activities of crude preparations of egg and oocyte RNA polymerases are tested in vitro with different DNA templates. In contrast to the situation in vivo, it is found that poly[d(A-T1 - d(A-T)] is as efficiently transcribed in vitro by oocyte polymerase as by egg polymerase. Additionally poly[d(A-T) - d(A-T)] is transcribed ten-fold more efficiently in vitro than calf thymus native DNA. When poly(dA) - poly(dT), poly(dA), phiX, T2, and calf thymus denatured DNA are tested in vitro, only calf thymus denatured DNA is transcribed to a significant extent. The above results are discussed in relation to the known synthetic activities of Xenopus eggs and oocytes.