Characterization of the components of reconstituted Saccharomyces cerevisiae RNA polymerase I transcription complexes.
We have reconstituted specific RNA polymerase I transcription from three partially purified chromatographic fractions (termed A, B, and C). Here, we present the chromatographic scheme and the initial biochemical characterization of these fractions. The A fraction contained the RNA polymerase I transcription factor(s), which was necessary and sufficient to form stable preinitiation complexes at the promoter. Of the three fractions, only fraction A contained a significant amount of the TATA binding factor. The B fraction contributed RNA polymerase I, and it contained an essential RNA polymerase I transcription factor that was specifically inactivated in response to a significant decrease in growth rate. The function of the C fraction remains unclear. This reconstituted transcription system provides a starting point for the biochemical dissection of the yeast RNA polymerase I transcription complex, thus allowing in vitro experiments designed to elucidate the molecular mechanisms controlling rRNA synthesis.
Riggs
D L
DL
Department of Botany and Microbiology, University of Oklahoma, Norman 73019.
Peterson
C L
CL
Wickham
J Q
JQ
Miller
L M
LM
Clarke
E M
EM
Crowell
J A
JA
Sergere
J C
JC
D002852:Chromatography, Ion Exchange; D004275:DNA, Ribosomal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D012318:RNA Polymerase I / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D016385:TATA Box; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D014461:Ultracentrifugation
D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D014157:Transcription Factors; D012318:RNA Polymerase I