After synthesis of short, nascent oligonucleotide in the presence of (32P)DNA, GTP, CTP, UTP and 3'dATP, one can excise with deoxyribonuclease a ternary complex of RNA polymerase, protected DNA and oligonucleotide, while the enzyme simply bound to the template is removed by increasing the ionic strength. This ternary complex is retained on nitrocellulose membranes. On polyacrylamide gel electrophoresis it migrates faster than RNA polymerase alone. The protected portion of the DNA is constituted of about 75 nucleotides. It might represent the sites for RNA initiation.

Journal Article↗

PubMed1968

[Iniatiation of RNA chains by RNA polymerase in vitro].

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Adenosine Triphosphate↗

PubMed1967

[Interaction between pigments and nucleic acids. 3. In vitro inhibition of the functioning of E. coli RNA polymerase by luteoskyrine].

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Adenosine Triphosphate↗

PubMedDate not supplied

Stimulation of transcription of the yeast tRNATyr gene in cell-free extracts by tyrosyl-tRNA synthetase.

Eukaryotic transfer RNA genes have two internal discontinuous control regions, the A and B blocks, which correspond approximately to the D-stem and the pseudo-U arm of tRNA. In reconstituted transcription systems at least two components are required to direct accurate initiation by RNA polymerase (refs 4,5). However, little is known about the mechanism of interaction of the internal promoter sequences with factors and RNA polymerase C within the transcription complex, although tRNA-like conformation of the B block sequence was surmised to be critical for DNA recognition. By analogy with the 5S RNA system, where a transcription factor required for 5S DNA expression was shown to interact both with 5S RNA and with the noncoding strand of the 5S gene, we explored the possibility that a protein which normally binds to tRNA could also interact with the tRNA gene and regulate its transcription. Here we show that in vitro transcription of the yeast SUP4 tRNATyr gene in crude yeast extracts is strongly stimulated by tyrosyl-tRNA synthetase (TyrRS) but not by two other non-cognate synthetases. Substrates of the synthetase, tRNATyr and tyrosine, interfere with stimulation of tRNA synthesis.

Amino Acyl-tRNA Synthetases↗

PubMedDate not supplied

Selective proteolysis defines two DNA binding domains in yeast transcription factor tau.

Transcription of eukaryotic transfer RNA genes involves, as a primary event, the stable binding of a protein factor to the intragenic promoter. The internal control region is composed of two non-contiguous conserved sequence elements, the A and B blocks. These are variably spaced depending on the genes. tau, a large transcription factor purified from yeast cells, interacts with these two control elements as shown by DNase I footprinting, exonuclease digestion, dimethyl sulphate protection experiments and by analysis of point mutations. Here we used a limited proteolysis treatment to obtain a smaller form of tau with drastically altered DNA binding properties. A protease-resistant domain interacts solely with the B block region of tRNA genes.

Binding Sites↗