Abstract
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.
Highlights
We have used an Eppendorf centrifuge for isolation defined the approximate orderof interaction of these factors of transcription complexes assembledon VARNAgenes with DNA templates and RNApolymerases [14,15,16,17,18,19]
Similar to the 5 S rRNA gene, sedimentable, stable transcription preinitiation complexes could be assembled from two VARNA genes,two EB virusspecific EBER genes, four human tRNAgenes, 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
For conclusive determination of the number of transcription factors involved in RNA polymerase III-mediated transcriptionand forrevealing the dynamicprocess of RNA polymerase I11 transcription machineries,we have chosen an alternative means which was t o biochemically characterize isolated transcription machineries assembled on either the plexes were stable at from 4 to 37 “C, and somewhat wild type or mutant VARNAl gene [16]
Summary
We have used an Eppendorf centrifuge for isolation defined the approximate orderof interaction of these factors of transcription complexes assembledon VARNAgenes with DNA templates and RNApolymerases [14,15,16,17,18,19]. Preparation and inVitro Transcription of Sedimentable Transcrip- GMPs [28], and in the absence of exogenously added tion Complexes-Preincubation of DNA with NTP-depleted extracts GTP, the endogenous GTPconcentration was too low to was usually done in a 0.05-ml reaction mixture under optimal conditions for formation of stable complexes a t 29 “C for 60 min unless indicated.
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