Histones are required for transcription of yeast rRNA genes by RNA polymerase I.
Nucleosomes and their histone components have generally been recognized to act negatively on transcription. However, purified upstream activating factor (UAF), a transcription initiation factor required for RNA polymerase (Pol) I transcription in Saccharomyces cerevisiae, contains histones H3 and H4 and four nonhistone protein subunits. Other studies have shown that histones H3 and H4 are associated with actively transcribed rRNA genes. To examine their functional role in Pol I transcription, we constructed yeast strains in which synthesis of H3 is achieved from the glucose-repressible GAL10 promoter. We found that partial depletion of H3 (approximately 50% depletion) resulted in a strong inhibition (>80%) of Pol I transcription. A combination of biochemical analysis and electron microscopic (EM) analysis of Miller chromatin spreads indicated that initiation and elongation steps and rRNA processing were compromised upon histone depletion. A clear decrease in relative amounts of UAF, presumably caused by reduced stability, was also observed under the conditions of H3 depletion. Therefore, the observed inhibition of initiation can be explained, in part, by the decrease in UAF concentration. In addition, the EM results suggested that the defects in rRNA transcript elongation and processing may be a result of loss of histones from rRNA genes rather than (or in addition to) an indirect consequence of effects of histone depletion on expression of other genes. Thus, these results show functional importance of histones associated with actively transcribed rRNA genes.
Department of Biological Chemistry, University of California, Irvine, CA 92697-1700, USA.
Prasad
P
Tongaonkar
Sarah L
SL
French
Melanie L
ML
Oakes
Loan
L
Vu
David A
DA
Schneider
Ann L
AL
Beyer
Masayasu
M
Nomura
D002843:Chromatin / Q000648:ultrastructure; D020459:Genes, rRNA / Q000235:genetics*; D006657:Histones / Q000378:metabolism*; D008854:Microscopy, Electron; D012318:RNA Polymerase I / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
D002843:Chromatin; D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C402483:upstream activation factor, S cerevisiae; D012318:RNA Polymerase I