Tipper DJ.Thiolutin
inhibits yeast ribonucleic
acid polymerases.
J Bacteriol. 1973 Oct;116(1):245-56.
Yeast ribonucleic acid (RNA) polymerase II, isolated after
fractionation on diethylaminoethyl (DEAE)-cellulose (DE-52) or
on DEAE-Sephadex (A-25), is 50% inhibited by 1.5 mug of
alpha-amanitin. This inhibition is independent of the sequence
of interaction of enzyme, template, nucleotides, and antibiotic
and is expressed immediately on addition of alpha-amanitin to a
preparation actively synthesizing RNA. Thus, alpha-amanitin's
primary effect is inhibition of elongation of preinitiated RNA
sequences in this system, as in others. A single peak of
alpha-amanitin-resistant RNA polymerase activity (I) was eluted
before enzyme II on either column. On A-25 but not on DE-52, a
third peak of activity (III) was eluted after enzyme II. This
activity was also resistant to alpha-amanitin. Enzymes I, II,
and III were 50% inhibited by 3, 4, and 3 mug of thiolutin per
ml, respectively. The extent of inhibition was independent of
the nature of the template (native or denatured salmon sperm
deoxyribonucleic acid or poly(dA-dT) or of the presence of 0.4
mM dithiothreitol, but this marked inhibition was only seen when
enzymes were preincubated with thiolutin in the absence of
template. Template protected the enzymes against thiolutin in
the absence of nucleotides. Either the sensitive site on the
polymerase is only accessible to thiolutin before interaction
with template or thiolutin inhibits functional
polymerase-template interaction but not elongation of
preinitiated RNA chains.
PMID: 4583213
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