Factors influencing promoter site selection by Escherichia coli RNA polymerase were investigated using T7 DNA as template. The utilization of the three major early promoters A1, A2 and A3, was followed by processing the RNA transcripts with RNAase III, which generates the three corresponding initiator RNA fragments. The three promoters proved to be functionally distinct. A strong differential effect of temperature and ionic strength on promoter selection was observed. The transition temperature of promoters A1, A2 and A3 was measured directly by preincubating, at different temperatures, RNA polymerase and T7 DNA, with primer-substrate combinations which selected each site independently. The transition temperature of the three sites was markedly different. Promoter A3 was used predominantly at low temperature, whereas A1 or A2 promoters were gradually activated by increasing the temperature. The temperature response curves were strongly dependent upon the salt concentration. On the other hand, challenge experiments with rifampicin or poly (inosinic acid) showed that, once preinitiation complexes are formed by incubating RNA polymerase with DNA at 37 degrees C, the three sites A1, A2 and A3 promote chain initiation with equal efficiency and at the same rate.

Binding Sites↗

PubMed1976

Two forms of RNA polymerase B in yeast. Proteolytic conversion in vitro of enzyme BI into BII.

Special care to prevent proteolysis during yeast RNA polymerase B purification leads to the appearance of two forms of enzymes, BI and BII, with different molecular weight (465 000) and 435 000, respectively). The two forms of enzyme can be separated by ion-exchange chromatography or polyacrylamide gel electrophoresis. Their subunit structures were compared by sodium dodecylsulfate gel electrophoresis, the only observed difference between the two enzymes is in the molecular weight of the heaviest subunit which is 220 000 for enzyme BI and 180 000 for enzyme BII. Otherwise, the two enzymes have seven common subunits of molecular weights 150 000, 45 000, 26 000, 22 500, 14 500, 12 500 and 9000. Two additional polypeptide chains of 32 000 and 16 500 Mr are dissociated from the enzyme upon polyacrylamide gel electrophoresis or DEAE Sephadex chromatography. The largest subunit of enzyme BI (Mr 220 000) can be specifically cleaved in vitro by a yeast protease extract, generating a polypeptide chain indistinguishable from the largest subunit of enzyme BII. This proteolytic cleavage of enzyme BI in vitro is inhibited by phenylmethylsulfonyl fluoride and does not significantly change the activity of the enzyme with single-stranded or double-stranded DNA as template. The precursor-product relationship of the different forms of class B RNA polymerases in eukaryotic cells is discussed.

Amanitins↗

PubMed1976

Structural studies on yeast RNA polymerases. Existence of common subunits in RNA polymerases A(I) and B(II).

The subunits of yeast RNA polymerases A(I) and B(II) were characterized using several techniques. The present studies demonstrate that the A and B enzymes possess three subunits, which are indistinguishable on the basis of molecular weight, isoelectric point, and fingerprint pattern. The three common subunits belong to the small molecular weight components of the enzymes. By polyacrylamide gel electrophoresis with sodium dodecyl sulfate they migrate with apparent molecular weights of 27,000, 23,000, and 14,500, respectively. A two-dimensional subunit mapping technique on polyacrylamide gel was used to separate the subunits according to isoelectric point and molecular weight. The common polypeptides co-migrated on three spots corresponding to isoelectric points of 9.2 (27,000), 4.5 (23,000), and 4.6 (14,500). The fingerprints of the 35S-labeled tryptic peptides of the presumptive common subunits were found to be essentially identical. Finally, the presence of common subunits was supported by the fact that antibodies against pure RNA polymerase A cross-react with and inhibit RNA polymerase B. Except for the common subunits, it is likely that RNA polymerases A and B are primarily made of distinct gene products for the following reasons. A total of 13 polypeptide chains are present in enzyme A, whereas 10 polypeptides are found in enzyme B. The molecular weight, isoelectric point, and sulfur content of the majority of these polypeptide chains are different in the two enzymes. No similarity was found in the 35S-peptide fingerprint from a number of A and B subunits of slightly different molecular weight. Finally, antibodies against the largest subunit from RNA polymerase A do not cross-react with or inhibit RNA polymerase B. The data are discussed in terms of structural organization of eukaryotic RNA polymerases.

DNA-Directed RNA Polymerases↗

PubMed1976

Further characterization of yeast RNA polymerases. Effect of subunits removal.

Two forms of yeast RNA polymerase A are resolved by phosphocellulose chromatography. One of these, called RNA polymerase A, is lacking two polypeptide chains of 48,000 and 37,000 daltons. The properties of the two enzymes are compared in the present paper. RNA polymerase A transcribes d(A-T)n with a similar efficiency as the complete enzyme, but it is comparatively much less active with native DNA. The two enzymes can also be differentiated on the basis of their ionic strength and divalent cation requirements. RNA polymerase A has a particularly low activity at high salt and low Mg2+ concentrations. Thermal inactivation curves of the two enzymes are different when residual activity is assayed with native DNA. In contrast with d(A-T)n as template the apparent inactivation curves of the two enzymes are identical. The data suggest that the two dissociable polypeptide chains play an important role in transcription. The template specificity of yeast RNA polymerase B was further investigated using SV40 DNA-FI as template. RNA polymerase B is able to retain [3H]SV40 DNA-FI on nitrocellulose filters but the enzyme-DNA complex is very unstable. The observation that RNA polymerase B can transcribe to some extent a supercoiled DNA but not a linear double stranded template supports the hypothesis that the enzyme needs some unpaired DNA structure to initiate transcription.

Cations, Divalent↗

PubMed1975

On the mode of action of lomofungin, an inhibitor of RNA synthesis in yeast.

Lomofungin is a potent inhibitor of RNA synthesis in yeast. Studies on the mode of action of the inhibitor were carried out using yeast RNA polymerases A and B and bacterial RNA polymerase. In vitro inhibition of RNA synthesis is independent of the nature and concentration of the template used and of the nucleoside triphosphate concentration. The extent of inhibition is strongly dependent upon the nature and concentration of divalent cations used to simulate transcription. The three RNA polymerases were inhibited to the same extent in the presence of Mn2+ ions whereas little inhibition was observed with Mg2+ ions. Spectrophotometric studies reveal the formation of different complexes between lomofungin and divalent cations (Mn2+, Mg2+, or Zn2+) with the respective stoichiometries of 0.5, 1, and 2 divalent cations per molecule of lomofungin. The complexes formed depend upon the nature of the divalent cation involved. No direct interaction between lomofungin and DNA could be observed in the presence of divalent cations but evidence is presented that lomofungin interacts with yeast RNA polymerase A. Inhibition of RNA synthesis occurs at the level of both chain initiation and elongation.

Chelating Agents↗

PubMed1975

Interaction of RNA polymerase from Escherichia coli with DNA. Analysis of T7 DNA early-promoter sites.

A method was devised for directing RNA polymerase on a single promoter site on T7 DNA. Initiation complexes were formed on each of the three main promoter sites using one dinucleotide plus one nucleoside triphosphate. The ternary initiation complexes are resistant to rifampicin action, to inhibition by (rI)n at 0 degrees C and are stable at high salt concentrations. A minimum of a trinucleotide is required to form a stable ternary complex. To determine which promoter site was selected by RNA polymerase during initiation, the (rI)n-resistant RNA was digested by RNAse III to generate three characteristic initiator RNA fragments, resolved by gel electrophoresis. The three major promoter sites could be selected individually by using different primer and substrate combinations ApC plus ATP selected promoter A3, CpG plus CTP selected A2 and CpC plus ATP specified preferentially A1. A number of primer-substrate combinations specified each site at low salt concentration but the substrate requirement became very stringent at high salt concentration, suggesting that the postulated local opening of the promoter site could be more or less extensive, depending on the ionic strength. The minimum opening observed at high salt concentration corresponded to the insertion of a leader trinucleotide sequence. The promoter region melted by RNA polymerase at low salt concentration was (G plus C)-rich and corresponded to about 9 to 11 base pairs. Sequences of the melting recognition regions were tentatively inferred from the results.

Coliphages↗

PubMed1975

Dissociation of two polypeptide chains from yeast RNA polymerase A.

Yeast RNA polymerase A (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) can be converted to a new form of enzyme, called RNA polymerase A*, which is lacking two polypeptide chains of 48,000 and 37,000 daltons. Apart from these two missing polypeptides the subunit structures of RNA polymerases A and A* are indistinguishable. RNA polymerase A* differs from the complete enzyme in its electrophoretic and chromatographic behavior, template requirements, and alpha-amanitin sensitivity. RNA polymerase A* transcribes the alternated copolymer d(A-T)n with the same efficiency as RNA polymerase A but its specific activity is greatly reduced with native calf thymus DNA as template. The transcription of a variety of synthetic templates is also altered by removal of the two polypeptide chains. RNA polymerase A* is inhibited by high concentrations of alpha-amanitin (500 mug/ml), whereas RNA polymerase A is comparatively less sensitive to the toxic peptide. The data are discussed in terms of possible roles of the two dissociable polypeptides.

Amanitins↗

PubMed1974