No abstract

Fractionation of yeast extracts on heparin-agarose revealed the presence of a DNA footprinting activity that interacted specifically with the 5'-upstream region of TEFI and TEF2 genes coding for the protein synthesis elongation factor EF-la, and of the ribosomal protein gene RPSIA.

The c-Myb protein is a sequence-specific DNA binding protein that activates transcription in hematopoietic cells. Three imperfect repeats (R1, R2, and R3) that contain regularly spaced tryptophan residues form the DNA binding domain of c-Myb. A fragment of c-Myb that contained the R2 and R3 regions bound specifically to a DNA sequence recognized by c-Myb plus ten additional base pairs at the 3' end of the element. The R2R3 fragment was predicted to contain two consecutive helix-turn-helix (HTH) motifs with unconventional turns. Mutagenesis of amino acids in R2R3 at positions that correspond to DNA-contacting amino acids in other HTH-containing proteins abolished specific DNA binding without affecting nonspecific DNA interactions.

A yeast extract was fractionated to resolve the factors involved in the transcription of yeast tRNA genes. An in vitro transcription system was reconstituted with two separate protein fractions and purified RNA polymerase C (III). Optimal conditions for tRNA synthesis have been determined. One essential component, termed tau factor, was partially purified by conventional chromatographic methods on heparin‐agarose and DEAE‐Sephadex; it sedimented as a large macromolecule in glycerol gradients (mol. wt. approximately 300 000). tau factor was found to form a stable complex with the tRNA gene in the absence of other transcriptional components. Complex formation is very fast, is not temperature dependent between 10 degrees C and 25 degrees C and does not require divalent cations. The factor‐DNA complex is stable for at least 30 min at high salt concentration (0.1 M ammonium sulfate). These results indicate that gene recognition by a specific factor is a primary event in tRNA synthesis.

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 aamanitin sensitivity. RNA polymerase A* transcribes the alternated copolymer d(A-T). 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 a-amanitin (500 Mg/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.There is currently considerable interest in the structural and functional properties of the multiple forms of RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) isolated from eukaryotic cells. Several laboratories have purified these enzymes from various organisms (1). Nuclear RNA polymerases. are quite complex multimeric proteins which appear to consist of two large subunits in equimolar amount associated with a series of smaller polypeptide chains (2-6). This structural complexity suggests the likelihood that each enzyme form is made of a fundamental enzyme surrounded with regulatory components or specificity determinants having a specialized role in transcription. Although no available evidence yet supports this hypothesis, past experience with other polymerizing enzymes shows that the basic templatedirected polymerization reaction can be carried out by relatively simple proteins (7-9). If small polypeptide chains present in the RNA polymerase molecule were indeed endowed with regulatory function one could expect some of them to be loosely and reversibly associated to the basic enzyme.As our experience with yeast RNA polymerase A accumulated, persistent peculiarities were noted during fractionation which indicated that the enzyme could be resolved into two distinct enzymatic fractions. It is the purpose of this report to detail the experiments which exploited these hints. The data show that two polypeptide chains of 48,000 and 37,000 daltons are reversibly associated to RNA polymerase A. Following gel electrophoresis or phosphocellulose chromatography RNA polymerase A can be converted to RNA polymerase A*, which is lacking these two satellite proteins. RNA-polymerase A* differs from the complete enzyme in its template requirements as well as other properties. A preliminary report of this work has been published (10). MATERIALS AND METHODSNucleic Acids and RNA Polymerases. Synthetic polymers w...