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Selective inactivation of two components of the multiprotein transcription factor TFIIIB in cycloheximide
G Dieci1, L Duimio, G Peracchia
1Institute of Biochemical Sciences, University of Parma, Italy.
Abstract:
Following protein synthesis inhibition in cycloheximide growth-arrested yeast cells, the rates of tRNA and 5 S RNA synthesis decrease with apparent half-times of about 20 and 10 min, respectively. This effect is mimicked by extracts of treated cells, and the impairment of tRNA gene transcription activity that is observed in vitro parallels the in vivo inactivation of RNA polymerase III transcription. As revealed by experiments in which partially purified class III transcription factors were singly added to extracts of treated cells, only the activity of the multiprotein transcription factor TFIIIB is severely impaired after 3 h of cycloheximide treatment. Similar assays carried out in an in vitro transcription system in which TFIIIB activity was reconstituted by a combination of the TATA box-binding protein (TBP), the 70-kDa component TFIIIB70, plus a partially purified fraction known as B" have shown that the latter two components are both necessary and sufficient to restore control levels of transcription. Their activity, but not TBP activity, is considerably reduced in extracts of treated cells. TFIIIB70 and a component of fraction B" thus appear to be the selective targets of the down-regulation of polymerase III transcription that is brought about by cycloheximide. A substantial depletion of the TFIIIB70 polypeptide was detected by Western immunoblot analysis of extracts derived from cycloheximide growth-arrested cells, indicating that the inactivation of this TFIIIB component results primarily from its enhanced destabilization under conditions of protein synthesis inhibition.
Insights
Protein synthesis inhibition in yeast reduces tRNA and 5S RNA synthesis by targeting transcription factor TFIIIB. Specifically, TFIIIB70 destabilization leads to RNA polymerase III transcription down-regulation.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Gene Transcription
Background:
- Protein synthesis inhibition affects cellular RNA production.
- RNA polymerase III is crucial for tRNA and 5S RNA synthesis.
- Transcription factor IIIB (TFIIIB) regulates RNA polymerase III activity.
Purpose of the Study:
- To investigate the molecular mechanisms underlying RNA polymerase III transcription down-regulation during protein synthesis inhibition in yeast.
- To identify the specific components of the transcription machinery affected by cycloheximide treatment.
Main Methods:
- Yeast cell culture and cycloheximide treatment.
- In vitro transcription assays using cell extracts and partially purified transcription factors.
- Western immunoblot analysis to detect protein levels.
Main Results:
- Cycloheximide treatment significantly impairs tRNA and 5S RNA synthesis rates in yeast.
- In vitro transcription assays revealed that only TFIIIB activity is severely affected.
- Reconstitution experiments showed that TFIIIB70 and a component of fraction B" are essential for restoring transcription, and their activity is reduced in treated cells.
- Western blot analysis confirmed a substantial depletion of the TFIIIB70 polypeptide.
Conclusions:
- TFIIIB70 and a component of fraction B" are the selective targets for cycloheximide-induced down-regulation of RNA polymerase III transcription.
- The inactivation of TFIIIB is primarily due to enhanced destabilization of TFIIIB70 under protein synthesis inhibition.