Active site labeling of the RNA polymerases A, B, and C from yeast

RNA polymerases A, B, and C from yeast were modified by reaction with 4-formylphenyl-gamma-ester of ATP as priming nucleotide followed by reduction with NaBH4. Upon phosphodiester bond formation with [alpha-32P]UTP, only the second largest subunit, A135, B150, or C128, was labeled in a template-depe...

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Bibliographic Details
Published in:The Journal of biological chemistry 1987-10, Vol.262 (30), p.14377-14380
Main Authors: Riva, M, Schäffner, A R, Sentenac, A, Hartmann, G R, Mustaev, A A, Zaychikov, E F, Grachev, M A
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - pharmacology
Affinity Labels
Amanitins - pharmacology
Analytical, structural and metabolic biochemistry
Binding Sites
Biological and medical sciences
DNA-Directed RNA Polymerases - analysis
Enzymes and enzyme inhibitors
ETIQUETADO
Fundamental and applied biological sciences. Psychology
LABELLING
LEVADURA
LEVURE
MARQUAGE
Oligonucleotides - analysis
RNA Polymerase I - analysis
RNA Polymerase II - analysis
RNA Polymerase III - analysis
RNA-directed RNA polymerase
Saccharomyces cerevisiae
TRANSFERASAS
TRANSFERASE
TRANSFERASES
YEASTS
Yeasts - enzymology
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Summary:RNA polymerases A, B, and C from yeast were modified by reaction with 4-formylphenyl-gamma-ester of ATP as priming nucleotide followed by reduction with NaBH4. Upon phosphodiester bond formation with [alpha-32P]UTP, only the second largest subunit, A135, B150, or C128, was labeled in a template-dependent reaction. This indicates that these polypeptide chains are functionally homologous. The product covalently bound to B150 subunit was found to consist of a mixture of ApU and a trinucleotide. Enzyme labeling exhibited the characteristic alpha-amanitin sensitivity reported for A and B RNA polymerases. Labeling of both large subunits of enzyme A and B but not of any of the smaller subunits was observed when the reduction step stabilizing the binding of the priming nucleotide was carried out after limited chain elongation. These results illustrate the conservative evolution of the active site of eukaryotic RNA polymerases.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(18)47803-9