ABSTRACT
There is limited information on how eukaryotic RNA polymerases (Pol) recognize their cognate preinitiation complex. We have characterized a polypeptide copurifying with yeast Pol III. This protein, C17, was found to be homologous to a mammalian protein described as a hormone receptor. Deletion of the corresponding gene, RPC17, was lethal and its regulated extinction caused a selective defect in transcription of class III genes in vivo. Two-hybrid and coimmunoprecipitation experiments indicated that C17 interacts with two Pol III subunits, one of which, C31, is important for the initiation reaction. C17 also interacted with TFIIIB70, the TFIIB-related component of TFIIIB. The interaction domain was found to be in the N-terminal, TFIIB-like half of TFIIIB70, downstream of the zinc ribbon and first imperfect repeat. Although Pol II similarly interacts with TFIIB, it is notable that C17 has no similarity to any Pol II subunit. The data indicate that C17 is a novel specific subunit of Pol III which participates together with C34 in the recruitment of Pol III by the preinitiation complex.
Subject(s)
RNA Polymerase III/chemistry , RNA Polymerase III/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Conserved Sequence/genetics , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Genes, Essential/genetics , Humans , Molecular Sequence Data , Molecular Weight , Mutation/genetics , Open Reading Frames/genetics , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Precipitin Tests , Protein Binding , RNA Polymerase III/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Transcription Factor TFIIIB , Transcription Factors/genetics , Two-Hybrid System TechniquesABSTRACT
A mutation in the structural gene coding for seryl-tRNA synthetase in temperature-sensitive Escherichia coli K28 has been reported to alter the level of enzyme expression at high temperature (R. J. Hill and W. Konigsberg, J. Bacteriol. 141:1163-1169, 1980). We identified this mutation as a C-->T transition in the first base of codon 386, resulting in a replacement of histidine by tyrosine. The steady-state levels of serS mRNA in K28 and in the wild-type strains are very similar. Pulse-chase labeling experiments show a difference in protein stability, but not one important enough to account for the temperature sensitivity of K28. The main reason for the temperature sensitivity of K28 appears to be the low level of specific activity of the mutant synthetase at nonpermissive temperature, not a decreased expression level. Spontaneous temperature-resistant revertants were selected which were found to have about a fivefold-higher level of SerRS than the K28 strain. We identified the mutation responsible for the reversion as being upstream from the -10 sequence in the promoter region. The steady-state levels of serS mRNA in the revertants are significantly higher than that in the parental strain.