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M R Paule

Publications and source records attributed to M R Paule.

47 records · Page 3Linked to original sources
PubMed1979

DNA-dependent RNA polymerase III from Acanthamoeba castellanii: comparison of the catalytic properties of the trophozoite and cyst enzymes.

DNA-dependent RNA polymerase III was partially purified from trophozoites and immature cysts of the small soil ameba, Acanthamoeba castellanii. In contrast to the active modulation of the variety of transfer RNA species which are transcribed during encystment, no difference was found in the chromatographic or catalytic properties of the enzyme responsible for their transcription.

Amanitins↗
PubMed1978

DNA-dependent RNA polymerase II from Acanthamoeba castellanii. Comparison of the catalytic properties and subunit architectures of the trophozoite and cyst enzymes.

The actively growing cells (trophozoites) of the amoeba Acanthamoeba castellanii were found to contain three or perhaps four different forms of class II DNA-dependent RNA polymerase (EC 2.7.7.6). The chromatographic and catalytic properties of all forms of the Acanthamoeba class II polymerases suggest them to be cognates of the class II polymerases previously reported. The predominant form was purified to near homogeneity and its subunit composition determined. Nine different polypeptides were found associated with the purified enzyme: 21 000; 185 000; 140 000; 70 000; 35 000; 21 000; 19 000; 18 500 and 16 200. These polypeptides were interpreted in terms of two class II RNA polymerases which differ in the molecular weight of their largest subunit. When A. castellanii is transferred to a medium lacking nutrients, the cells undergo cellular differentiation resulting in the formation of metabolically inactive cells (cyst formation). During this process there are significant changes in the RNA sequences transcribed. In contrast to this, we find that the chromatographic and catalytic properties of all of the class II RNA polymerases remain unchanged. Further, the subunit architecture of the predominant form(s) of polymerase II is unaltered. These findings suggest that although new RNA sequences are transcribed during encystment their appearance is not a consequence of extensive alterations in the subunit composition of the major class II RNA polymerase.

Amanitins↗
PubMed1978

DNA-dependent RNA polymerases from Acanthamoeba castellanii. Multiple forms of the class III enzyme and levels of activity of the polymerase classes during encystment.

Multiple forms of class III DNA-dependent RNA polymerase have been found in a number of higher eukaryotic cells types (Chambon, P. (1975) Annu. Rev. Biochem. 44, 613--638). Similar multiple forms are reported here from a lower eukaryote, the soil amoeba Acanthamoeba castellanii. The levels of activity of all three RNA polymerase classes in whole cell extracts of Acanthamoeba during cellular differentiation were examined. In contrast to our previous observation (Detke, S. and Paule, M.R. (1975) Biochim. Biophys. Acta 383, 67--77) that the amount of the alpha-amanitin-sensitive polymerases (II + III) increase in the nucleus during encystment we find that the amount of polymerases II and III when solubilized from whole cells remains constant. The level of activity of polymerase I, however, remains constant both in nuclei and in whole cell extracts. Thus, the shutdown of ribosomal RNA synthesis occurring during encystment is not due to a decrease in the number of polymerase I molecules in the cell.

Amanitins↗
PubMed1975

DNA-dependent RNA polymerases from Acanthamoeba castellanii: properties and levels of activity during encystment.

Three DNA-dependent RNA polymerases have been isolated and partially purified from trophozoites of Acanthamoeba castellanii. Separated by DEAE-Sephadex chromatography, they have been designated polymerases, I, IIa and IIB according to their alpha-amanitin sensitivity and kinetic properties. I is completely insensitive to alpha-amanitin. IIa and IIb are sensitive to low concentrations (0.1 mug/ml) of alpha-amanitin; however, in order to achieve 100% inhibition much higher concentrations (130 mug/ml) are needed. Both I and II (a or b) have rather broad ionic strength optima (0.06--0.10 M (NH4)2SO4). All three prefer denatured over native DNA (I, 4:1; II, 2:1). Polymerase I utilizes magnesium better than manganese as divalent cation whereas II prefers manganese. When Acanthamoeba is transferred to a medium lacking nutrients, the cells undergo a synchronous differentiation resulting in cyst formation. In general agreement with the decrease in the rate of synthesis of its product (rRNA), the amount of polymerase I decreases relative to the amanitin sensitive polymerase(s). However, the absolute amount of polymerase I does not change. Rather, the levels of the amanitin sensitive enzymes increase during the first 10 h of encystment. Since the overall RNA synthesis rate decreases, these results suggest that the transcription rate is not controlled by specific enzyme levels alone.

Amanitins↗
PubMedDate not supplied

Initiation and regulation mechanisms of ribosomal RNA transcription in the eukaryote Acanthamoeba castellanii.

Acanthamoeba rRNA transcription involves the binding of a transcription initiation factor (TIF) to the core promoter of rDNA to form the preinitiation complex. This complex is formed in the absence of RNA polymerase I, and persists for multiple rounds of initiation. Polymerase I next binds to form the initiation complex. This binding is DNA sequence-independent, and is directed by protein-protein contacts with TIF. DNA melting occurs in a separate step. In contrast to most prokaryotic transcription, melting occurs only following nucleotide addition and beta-gamma hydrolysis of ATP is not required as for polymerase II. Growth-dependent regulation of rRNA transcription is accomplished by modification of RNA polymerase I. The inactive form of polymerase (PolE) is unable to bind to the promoter and has altered heat stability. PolE is still active in elongation; thus, the modification affects the polymerase site involved in TIF contact. Modification of a polymerases I and III common subunit has been detected leading to the suggestion that transcription of stable RNAs of the ribosome might be co-regulated by this mechanism.

Acanthamoeba↗