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PMID: 9580684 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

A differential response of wild type and mutant promoters to TFIIIB70 overexpression in vivo and in vitro.

Nucleic acids research ·Vol. 26 ·No. 10 ·1998-05-15 ·Pages 2344-52

Sethy-Coraci I, Moir RD, López-de-León A, Willis IM

Abstract

TFIIIB, the initiation factor for transcription by RNA polymerase III (pol III) is, in yeast, composed of three subunits: TBP, TFIIIB70/Brf1 and TFIIIB90. To determine the extent to which each of these subunits is limiting for pol III transcription, the effect of overexpressing each subunit was assessed on the expression of wild-type and promoter mutant pol III genes both in vivo and in vitro . In vivo , we find that the synthesis of wild-type pol III genes is not limited to a significant extent by the level of any TFIIIB subunit. There is, however, a two-fold increase in the synthesis of the promoter mutant gene, sup9-e A19-supS1 , in strains overexpressing TFIIIB70. The findings suggest that overexpression of TFIIIB70has a differential effect on the expression of pol III genes with strong versus weak promoters. In vitro transcription assays support this conclusion and reveal an inverse correlation between the transcriptional response to TFIIIB70overexpression and promoter strength. The individual TFIIIB subunits are nuclear by immunofluorescence and are calculated to have nuclear concentrations in the low micromolar range. In comparison, the factors are diluted 100-fold or more in whole cell extracts. This dilution accounts for the generally limiting nature of TFIIIB70in pol III gene transcription in vitro.

MeSH Terms
Cell Nucleus/chemistry DNA-Binding Proteins/analysis Gene Expression Regulation, Fungal/genetics Mutation Promoter Regions, Genetic/genetics RNA Polymerase III/metabolism RNA Precursors/biosynthesis RNA, Fungal/biosynthesis Saccharomyces cerevisiae/genetics Suppression, Genetic TATA-Box Binding Protein Transcription Factor TFIIB Transcription Factors/analysis,genetics,metabolism
Chemicals
DNA-Binding Proteins RNA Precursors RNA, Fungal TATA-Box Binding Protein Transcription Factor TFIIB Transcription Factors RNA Polymerase III
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Sethy-Coraci I
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Moir R D
López-de-León A
Willis I M
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/26.10.2344
Published
1998-05-15
Pages
2344-52
Language
English
Region
England
NLM ID
0411011
PMCID
PMC147558
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/26.10.2344 PMC Full Text View journal details
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