Large scale isolation of RNA polymerase I of Tetrahymena pyriformis and the identification of polymerase-associated polypeptides

A procedure has been developed for a large scale and rapid isolation of RNA polymerase I (EC 2.7.7.6) of Tetrahymena pyriformis. The enzyme is precipitated from the cell homogenate by Polymin P, extracted from the sediment and separated from RNA polymerase II by a treatment with phosphocellulose. The further purification procedure involves sedimentation in glycerol gradients and chromatography on heparin-Sepharose and DEAE-Sephadex. The last step achieved the separation of RNA polymerase I from RNA polymerase III. On the basis of different criteria RNA polymerase I is assumed to consist of two large subunits of 180 and 118 kDa and nine subunits smaller than 50 kDa. Additional polypeptides have been identified which are associated with RNA polymerase I but are not found in integral stoichiometric amounts. Except for certain minor differences RNA polymerase I purified from the cell homogenate shows the same structure as the enzyme obtained from isolated macronuclei (Mueller et al., 1985).     

A novel adenosine triphosphatase isolated from RNA polymerase preparations of Escherichia coli. II. Enzymatic properties and molecular structure.

An adenosinetriphosphatase (ATPase) [EC 3.6.1.3] copurified with the DNA-dependent RNA polymerase [EC 2.7.7.6] from Escherichia coli was isolated to apparent homogeneity and some of its functional as well as structural properties were examined. Although the novel ATPase exhibited metal requirements similar to those of Mg2+, Ca2+-ATPase, its response to NaN3 and antisera appeared completely different from that of the Mg2+, Ca2+-ATPase. The purified ATPase was found to be a large protein with a molecular weight of 9.3X10(5) daltons, composed of identical subunits of 7X10(4) daltons. When viewed under an electron microscope, the ATPase appeared to be very similar to material previously misidentified as the RNA polymerase. The physiological role of the novel ATPase, however, remains unclear.     

Identification of subunits of gonococcal RNA polymerase by immunoblot analysis: evidence for multiple sigma factors.

Heparin-agarose and single-stranded DNA-cellulose chromatography were used to purify RNA polymerase 25-fold from Neisseria gonorrhoeae, and the activity of the polymerase was characterized in altered assay systems. The core subunits (beta, beta', and alpha) were tentatively identified as major proteins copurifying with polymerase activity. The identification of the core subunits was confirmed by Western (immunoblot) analysis with polyclonal antisera to Escherichia coli core RNA polymerase. Gonococcal sigma factor heterogeneity was examined by Western blot analysis with polyclonal antiserum to the major E. coli sigma factor, sigma 70, to the E. coli heat shock sigma factor, sigma 32, and with a monoclonal antiserum to Salmonella typhimurium NtrA (sigma 54). Purified RNA polymerase and whole-cell extracts from type 1, type 4, heat-shocked, and anaerobically grown gonococci were examined. Four putative gonococcal sigma factors were detected in purified RNA polymerase preparations and in whole-cell extracts from all cell types. Two of these bands appeared as a doublet, which had an estimated Mr of 80,000. A single lower-Mr band, estimated to be 40,000, was also present. All three of these bands reacted with antisera to E. coli sigma 70 and to E. coli sigma 32. A fourth gonococcal protein reacted solely with a highly specific monoclonal antibody to sigma 54 and had an Mr of 90,000. We conclude that N. gonorrhoeae may contain multiple sigma factors, which it may use to regulate gene expression.     

Subunit assembly and metabolic stability of E. coli RNA polymerase

Immunological cross-reaction was employed for identification of proteolytic fragments of E. coli RNA polymerase generated both in vitro and in vivo. Several species of partially denatured but assembled RNA polymerase were isolated, which were composed of fragments of the two large subunits, beta and beta', and the two small and intact subunits, alpha and sigma. Comparison of the rate and pathway of proteolytic cleavage in vitro of unassembled subunits, subassemblies, and intact enzymes indicated that the susceptibility of RNA polymerase subunits to proteolytic degradation was dependent on the assembly state. Using this method, degradation in vivo was found for some, but not all, of the amber fragments of beta subunit in merodiploid cells carrying both wild-type and mutant rpoB genes. Although the RNA polymerase is a metabolically stable component in exponentially growing cells of E. coli, degradation of the full-sized subunits was found in two cases, i.e., several temperature-sensitive E. coli mutants with a defect in the assembly of RNA polymerase and the stationary-phase cells of a wild-type E. coli. The in vivo degradation of RNA polymerase was indicated to be initiated by alteration of the enzyme structure.     

Identification of two different RNase H activities associated with yeast RNA polymerase A.

Two ribonuclease H activities have been found in yeast RNA polymerase A. The nuclease activities comigrated with subunits A49 (Mr = 49,000) and A40 (Mr = 40,000), after electrophoresis in a sodium dodecyl sulfate polyacrylamide gel containing [32P](rG)n . (dC)n as substrate. Both activities were also found, among other nucleases, in a high salt chromatin extract. Several lines of evidence suggest that the chromatin RNase H of 49,000 daltons (RNase H49) is the same protein as subunit A49. They co-migrate on sodium dodecyl sulfate-gel electrophoresis, have the same chromatographic properties, and dissociate simultaneously from RNA polymerase A. Fractions containing RNase H49 stimulate RNA synthesis by RNA polymerase A* lacking A49 and A34.5 subunits. Finally, limited proteolysis of the protein band having RNase H49 activity yields the characteristic fingerprint of the A49 subunit. This subunit, therefore, exists in two states: bound to chromatin and associated with RNA polymerase A. On the other hand, it is not yet clear whether the RNase H activity of 40,000 daltons, associated with RNA polymerase A, is due to the A40 subunit or whether it represents a trace contamination by a very active nuclease tightly bound to the enzyme.     

Purification and immunological analysis of RNA polymerase II from Caenorhabditis elegans

We describe a rapid procedure for obtaining highly purified RNA polymerase II from the nematode Caenorhabditis elegans. The structure of the enzyme was examined by denaturing gel electrophoresis and found to consist of three large polypeptides (molecular weights 200,000, 175,000, and 135,000) and eight smaller polypeptides (molecular weights 29,500, 20,000, 16,000, 15,000, 13,000, 11,500, 10,500, and 9,500). As observed for the analogous enzyme from other organisms, the 175,000 polypeptide (II175) appeared to be a degraded form of the 200,000 polypeptide (II200). The structure of nematode RNA polymerase II closely resembles that of the corresponding enzyme from other animals. Four of its larger subunits shared antigenicity with Drosophila RNA polymerase II. Antibody raised against purified RNA polymerase II reacted with several enzyme subunits in "Western" blots of purified polymerase and impure enzyme fractions. Immunofluorescence staining was used to visualize RNA polymerase II in the nuclei of a nematode squash preparation and the nucleoplasm of cultured mammalian cells.     

Effect of temperature on nuclear membranes and nucleo-cytoplasmic RNA-transport in Tetrahymena grown at different temperatures.

The effect of temperature on the nuclear envelope structure and the transport of total RNA and ribosomal subunits from nucleus to cytoplasm was examined in Tetrahymena cells propagated at two different temperatures. Freeze-etch electron microscopy of cells grown at 23 and 18 degrees C detects the emergence of smooth areas on the fracture faces of the nuclear membranes upon lowering the temperature below approximately 15 and approximately 12 degrees C, respectively. Coincident with these freeze-etch changes, a discontinuous decrease is observed in the nucleocytoplasmic RNA-transport; this is probably not due to a cease in RNA-synthesis. Below the thermotropic discontinuity observed in the transport of total RNA in 18 degrees-cells the nucleocytoplasmic transport of the small and large ribosomal subunits is equally retarded. Recent temperature studies on the endoplasmic reticulum membranes of Tetrahymena suggest that the freeze-etch changes in the nuclear membranes are induced by a thermotropic clustering of the membrane lipids. We conclude that this lipid clustering induces the permanent protein constituents in the nuclear envelope pore complexes to change from a relatively "open" into a relatively "closed" state thus causing the observed decrease in RNA-transport.     

Identification of homologous ribosomal proteins in HeLa cells and in Tetrahymena pyriformis. A study of proteins binding 5-S RNA and acidic proteins released from 40-S subunits by EDTA

Tetrahymena pyriformis 60-S ribosomal subunits treated with EDTA release a 7-S particle containing 5-S RNA and a 36000-Mr protein that is similar to mammalian 5-S-RNA-binding protein L5 in molecular weight, in two-dimensional acrylamide gel mobility, and in peptide pattern as generated by a simple, one-dimensional acrylamide gel technique. Human and T. pyriformis 40-S ribosomal subunits, treated with buffers lacking magnesium or containing EDTA, release varying amounts of two large acidic proteins. We have identified these released proteins by two-dimensional gel electrophoresis.     

RPC19, the gene for a subunit common to yeast RNA polymerases A (I) and C (III)

Yeast RNA polymerases A (I) and C (III) share a subunit called AC19. The gene encoding AC19 has been isolated from yeast genomic DNA using oligonucleotide probes deduced from peptide sequences of the isolated subunit. This gene (RPC19) contains an intron-free open reading frame of 143 amino acid residues. RPC19 is a single copy gene that maps on chromosome II and is essential for cell viability. The amino acid sequence contains a sequence motif common to the Escherichia coli RNA polymerase alpha subunit, the Saccharomyces cerevisiae AC40 and B44.5 subunits, the human hRPB33 product, and the CnjC conjugation-specific gene product of Tetrahymena. The 5'-upstream region contains a sequence element, the PAC box, that has been conserved in at least 10 genes encoding subunits of RNA polymerases A and C.     

Purification of the DNA-dependent RNA polymerase from the myxobacterium Stigmatella aurantiaca.

The DNA-dependent RNA polymerase (EC 2.7.7.6) of the myxobacterium Stigmatella aurantiaca has been purified. It shows three main polypeptide bands with apparent molecular weights of 146,000, 105,000, and 40,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. beta and beta' subunits of the S. aurantiaca polymerase were shown to migrate in the 146,000-molecular-weight polypeptide band and the main sigma factor was shown to migrate in the 105,000-molecular-weight band by using heterologous antisera.     

Isolation and characterization of DNA-dependent RNA polymerase I of Tetrahymena pyriformis

A procedure for the separation and purification of DNA-dependent RNA polymerases [EC 2.7.7.6] from macronuclei of Tetrahymena pyriformis is described. We have used it to isolate and characterize the class I enzyme. RNA polymerase I was identified by its resistance against alpha-amanitin and its location in nucleoli. The purified enzyme consists of at least 12 major subunits with approximate molecular weights of 180,000, 118,000, 37,500, 36,000, 29,000, 27,500, 20,000, 18,500, 15,600, 14,500, 13,500, and 12,600. Chromatography on DEAE-Sephadex separated two forms of RNA polymerase I which differed in the presence of an additional polypeptide of 25 kDa. Independently of this polypeptide, the enzyme was found to segregate on DNA cellulose into a binding and a non-binding fraction. This type of heterogeneity was found to be unrelated to differences in molar ratios or molecular weights of the enzyme subunits. The catalytic properties of all enzyme subfractions were very similar and complied with the general characteristics of RNA polymerase I [cf. Roeder, R.G. (1976) in RNA Polymerase (Losick, R. & Chamberlin, M., eds.) pp. 285-329, Cold Spring Harbor Publ. Co., New York].     

Subunit topography of RNA polymerase (E. coli) in the complex with DNA.

E. Coli RNA polymerase binding to different DNAs (from E. Coli, 5-bromodeoxyuridine (BrdUrd) substituted DNA and poly [d(BrU-A)] was induced with ultraviolet (U.V.) light to form protein-DNA crosslinked complexes. Two independent methods of analysis, polyacrylamide gel electrophoresis in SDS and chloroform extraction indicated the formation of a stable complex between the enzyme and DNA. The complexes were formed under different ionic strength conditions, at low enzyme to DNA ratios in order to approach the conditions of specific binding. In contrast there was no crosslinking of the complex in 1 M KCl solution which dissociates the enzyme from DNA. The efficiency of formation of strongly bound complex was found to be much higher with holoenzyme than with core enzyme. The following results were obtained : 1) The large subunits beta and beta' were found to be bound to DNA. 2) Relatively small amount of sigma subunit were bound to DNA while alpha subunits were essentially not attached to DNA. The high binding affinity of beta and beta' subunits was also observed in the studies of isolated subunits. These results lead to a model of enzyme-DNA complex in which the large beta and beta' subunits provide the contacts between the RNA polymerase and the DNA.     

Studies on the functions of the RNA polymerase components by means of mutations

Summary It had been shown earlier, that RNA polymerase 13 S particles contain the large components with a molecular weight of about 3–10⁵ and small subunits with a molecular weight of 4·10⁴-1·10⁵. These polymerase components easily dissociate and reassociate with restoration of the enzyme activity.Both temperature-sensitive (tsX) and rifamycin-resistant (rif-r-I) mutations proved to affect the large polymerase component without changing the small subunits. These mutations were mapped at different, though closely linked, loci of metB-thi region of E. coli K12 chromosome. These results as well as certain literature data allow to conclude that the large RNA polymerase component consists of at least two polypeptides, one being altered by ts mutation, and the other—by rif-r mutation.The large polymerase component when separated from the small subunits retain the ability to bind to T2 phage DNA while the separate small subunits lack this property. Rifamycin does not affect RNA polymerase-T2 DNA binding while ts mutation leads to inability of the enzyme to form stable complexes with DNA. Therefore, it is likely that the polypeptide affected by ts mutation is responsible for the attachment of RNA polymerase to specific sites of DNA template. On the other hand, the small subunits as well as polypeptide of the large component, which determines RNA polymerase sensitivity to rifamycin, seem not to participate in the enzyme binding to DNA template. It is suggested, that the catalytic site of RNA polymerase is located in the large component and formed by rifamycin-binding polypeptide. The small subunits are supposed to have regulatory function and activate the large components.     

The role of subunits in yeast DNA-dependent ribonucleic acid polymerase A.

The properties of RNA polymerase A, which lacked the subunits of 48 000, 37 000 and 16 000 mol. wt., were compared with those of RNA polymerase A by using native calf thymus DNA as the template. The results showed that: (1) the specific activity of RNA polymerase A was about one-third that of RNA polymerase A; (2) more than 80% of RNA polymerase A, but only about 25% of RNA polymerase A, made RNA; (3) initiation by RNA polymerase A, but not by RNA polymerase A, began after a lag of 2 min; (4) the temperature-dependence for productive binding to DNA was greater for RNA polymerase A; (5) the apparent Km for UTP was greater for RNA polymerase A. These results support the supposition that the subunits missing from RNA polymerase A are involved in DNA binding [Huet, Dezélée, Iborra, Buhler, Sentenac & Fromageot (1976) Biochimie 58, 71-80] and show also that the loss of these subunits affects the elongation reaction.     

African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases.

A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli. After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced. The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases. The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit. Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV. ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication.