J. Biol. Chem., Vol. 264, Issue 17, 9753-9761, 06, 1989
Formation of large, sedimentable transcription complexes with VARNA genes and other related genes
GJ Wu
Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322.
We have used an Eppendorf centrifuge for isolation of transcription
complexes assembled on VARNA genes and other related genes with NTP-
depleted cell-free extracts. Similar to the 5 S rRNA gene, sedimentable,
stable transcription preinitiation complexes could be assembled from two
VARNA genes, two EB virus-specific EBER genes, four human tRNA genes, and
one human Alu-family RNA gene, suggesting that the 5 S rRNA-specific
transcription factor, TFIIIA, was not required for formation of these
sedimentable, stable preinitiation complexes. Parameters affecting assembly
of these complexes were sequences in circular DNA templates, sizes and
sequences of linear DNA templates, temperature and incubation time. These
complexes were stable at from 4 to 37 degrees C, and somewhat stable to
salt wash. From results of effects of various mutations on assembly of
these sedimentable complexes, we concluded that they were transcription
machineries. Addition of the supernatant and partially purified factors to
salt- washed complexes stimulated their transcription, we concluded that
these sedimentable complexes were minimal transcription machineries
containing suboptimal quantities of loosely bound transcription factors,
TFIIIB, and RNA polymerase III. DNase 1 footprints of these sedimentable
preinitiation complexes showed that two regions were protected, from +34 to
+80 including the B block promoter element, and from +98 to +105. Similar
DNase 1 footprints were also obtained from salt-washed complexes and stable
preinitiation complexes isolated by molecular sieve column chromatography.
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