Biomedical subjects

M C Schmidt

Publications and source records attributed to M C Schmidt.

At least 19 recordsLinked to original sources

PubMed1991

Requirement for acidic amino acid residues immediately N-terminal to the conserved domain of Saccharomyces cerevisiae TFIID.

TFIID binds to TATA boxes and initiates the assembly of general transcription factors and pol II on promoters. TFIID proteins from various species consist of a highly conserved carboxy terminal domain and very divergent amino terminal domains. We investigated the function of the non-conserved amino terminal domain (residues 1-60) of Saccharomyces cerevisiae TFIID (YIID, 240 residues) by testing the ability of a series of YIID amino terminal deletion mutants to complement a YIID deficient yeast strain. Mutants with deletions up to amino acid 48 restored the YIID deficient yeast strain to an apparently wild type phenotype. However, deletion up to position 57 or 60 produced yeast strains which formed extremely small colonies. Moreover, overexpression of YIID delta 2-57 or YIID delta 3-60 protein in the presence of wild type YIID resulted in a dominant-negative inhibition of growth. No difference between the basal transcriptional activity of wild type YIID and these amino terminal deletion mutants was observed in vitro. However, transcriptional activation in vivo of promoter-lacZ fusions showed that the YIID delta 2-57 deletion affects the ability of certain promoters (CUP1 and an HSP UAS-CYC1 promoter hybrid promoter) to respond to upstream factor stimulation. At least one inducible promoter, PHO5, was not affected by this deletion. The defect produced by YIID delta 2-57 was due to the deletion of several acidic residues present between residues 48 and 57. The results show that the conserved carboxy terminal domain of YIID is sufficient for cell viability. However, an acidic region just amino terminal to the conserved domain is required for normal growth and transcription control in most yeast strains.

Amino Acid Sequence↗

PubMed1991

Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change.

Transcription factor IID from Saccharomyces cerevisiae (YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0 degrees C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.

Binding Sites↗

PubMed1990

Cloning of a transcriptionally active human TATA binding factor.

Transcription factor IID (TFIID) binds to the TATA box promoter element and regulates the expression of most eukaryotic genes transcribed by RNA polymerase II. Complementary DNA (cDNA) encoding a human TFIID protein has been cloned. The human TFIID polypeptide has 339 amino acids and a molecular size of 37,745 daltons. The carboxyl-terminal 181 amino acids of the human TFIID protein shares 80% identity with the TFIID protein from Saccharomyces cerevisiae. The amino terminus contains an unusual repeat of 38 consecutive glutamine residues and an X-Thr-Pro repeat. Expression of DNA in reticulocyte lysates or in Escherichia coli yielded a protein that was competent for both DNA binding and transcription activation.

Amino Acid Sequence↗

PubMed1990

Transcriptional activation by Sp1 as directed through TATA or initiator: specific requirement for mammalian transcription factor IID.

Transcription of mammalian genes by RNA polymerase II often begins at a specific nucleotide, whose location is determined either by an upstream DNA element known as a TATA box or by an element positioned at the transcription start site called an initiator (Inr). By in vitro analysis of synthetic promoters, we demonstrate here that the TATA and Inr elements are functionally similar and that the Inr is contained between nucleotides -3 and +5 relative to the initiation site. Moreover, we found that a mammalian transcription factor IID (TFIID) protein fraction is required for transcriptional stimulation by an Sp1-dependent activating element placed upstream of either TATA or Inr elements. However, in these assays, the yeast TATA-binding protein, which previously was shown to function similarly to mammalian TFIID, could not efficiently substitute for the mammalian TFIID fraction. These results demonstrate that mammalian TFIID is functionally distinct from the yeast TATA-binding protein and may contain additional subunits or domains that are important for transcriptional activation from some promoters.

Adenovirus Early Proteins↗

PubMed1989

Yeast TATA-box transcription factor gene.

The first step in the transcription of most protein-encoding genes in eukaryotes is the binding of a transcription factor to the TATA-box promoter element. This TATA-box transcription factor was purified from extracts of the yeast Saccharomyces cerevisiae by using reconstitution of in vitro transcription reactions as an assay. The activity copurified with a protein whose sodium dodecyl sulfate/polyacrylamide gel mobility is 25 kDa. The sequence of the amino-terminal 21 residues of this protein was determined by sequential Edman degradation. A yeast genomic library was screened with mixed oligonucleotides encoding six residues of the protein sequence. The yeast TATA-box factor gene was cloned, and DNA sequencing revealed a 720-base-pair open reading frame encoding a 27,016-Da protein. The identity of the clone was confirmed by expressing the gene in Escherichia coli and detecting TATA-box factor DNA binding and transcriptional activities in extracts of the recombinant E. coli. The TATA-box factor gene was mapped to chromosome five of S. cerevisiae. RNA blot hybridization and nuclease S1 analysis indicated that the major TATA-box factor mRNA is 1.3 kilobases, including an unusually long 5' untranslated region of 188 +/- 5 nucleotides. Homology searches showed a region of distant similarity to the calcium-binding structures of calpains, a structure that has a conformation similar to the helix-turn-helix motif of DNA binding proteins.

Amino Acid Sequence↗

PubMed1989

Sp1 activates transcription without enhancing DNA-binding activity of the TATA box factor.

We have studied the interactions of the Sp1 and IID transcription factors with a simple RNA polymerase II promoter. The adenovirus E1B core promoter consists essentially of a GC box and a TATA box, binding sites for the Sp1 and IID transcription factors, respectively. The E1B promoter is accurately transcribed in vitro using a mammalian transcription system. Sp1 activates E1B transcription in vitro in reactions using IID factor isolated from either human or yeast cells. In DNase I footprinting studies, Sp1 bound rapidly to its recognition sequence even at 0 degrees C (t1/2 less than 1 min). In contrast, yeast IID bound more slowly (t1/2 approximately 6 min at 25 degrees C) and required thermal energy for stable binding to the TATA box sequence. Dissociation rates were measured by the addition of specific oligonucleotide competitors to preformed DNA-protein complexes. Sp1 dissociates rapidly (t1/2 less than 1 min) at 25 degrees C, while yeast IID dissociates with an estimated t1/2 of 1 h at 25 degrees C. Sp1 and yeast IID bound to the E1B promoter simultaneously but independently. The rates of binding and dissociation of these factors were not significantly affected by the presence of the other factor. Bound Sp1 factor did not alter or enhance the yeast IID footprint. Oligonucleotide challenge of in vitro transcription reactions indicated that Sp1 also did not enhance the binding of the human IID factor to the E1B promoter. Thus the Sp1 factor activates transcription of the E1B gene by a mechanism that does not enhance the DNA-binding activity of the IID factor. Sp1 factor activates E1B transcription by 5- to 10-fold in vitro. Under these in vitro transcription conditions, transcripts due to reinitiation from an individual promoter complex contribute only a small portion of the total yield of E1B transcripts. Thus Sp1 cannot activate transcription by increasing the rate of initiation events per complex. Instead it appears that Sp1 acts by increasing the number of productive transcription complexes formed in vitro.

Adenoviridae↗

PubMed1987

nusA protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites.

We have studied the factors that affect transcription termination in vitro at the tR2 terminator of bacteriophage lambda and at the T1 terminator of the Escherichia coli rrnB operon. Termination efficiency at both of these sites is enhanced by the E. coli nusA protein, giving final efficiencies of termination in vitro comparable to those estimated in vivo. Transcripts terminated in the presence of nusA protein are all released from the RNA polymerase complex, indicating that a complete termination reaction is involved, rather than simply induction of a long pause at the terminator. The termination factor activity of the nusA protein does not depend on the presence of rho protein and is not detectably enhanced by that factor. Thus, the nusA protein appears to play a pleiotropic role in E. coli transcription, serving as an antitermination factor, RNA polymerase subunit and true termination factor for some terminator sites.

Bacterial Proteins↗

PubMed1986

Nonenzymatic radiolabeling of protein by 32P-containing nucleotides.

We report a nonenzymatic reaction which results in the radiolabeling of proteins by 32P-containing nucleoside triphosphates. The labeling reaction does not require any cofactors, but is greatly enhanced by the presence of alcohols. Even under optimal conditions, less than 1% of the protein molecules undergo modification. This nonspecific labeling represents a serious artifact which may become significant in systems involving low levels of specific labeling, such as photoaffinity labeling. Since the reaction is not limited to specific proteins, this may, however, provide a simple and rapid procedure for the preparation of labeled proteins.

Adenosine Triphosphate↗

PubMed1984

Binding of rho factor to Escherichia coli RNA polymerase mediated by nusA protein.

The E. coli transcription termination factor rho binds specifically to purified nusA protein. Since nusA protein binds tightly to RNA polymerase, this provides a way of coupling rho to the elongating RNA polymerase complex through protein-protein interactions. These rho-nusA interactions may play a role in modulating rho action at certain terminators and could also be important in the action of antitermination factors such as lambda N protein.

Adenosine Triphosphatases↗

PubMed1984

Amplification and isolation of Escherichia coli nusA protein and studies of its effects on in vitro RNA chain elongation.

The Escherichia coli nusA gene product is an RNA polymerase binding protein which has been implicated in a variety of cellular and viral termination and antitermination processes. To facilitate large-scale preparation and biochemical studies of the nusA protein, we have cloned the nusA gene into a lambda PL-derived overexpression vector. E. coli strains bearing the resulting plasmid (pMS7) produce large amounts of nusA protein when induced, and the protein is easily purified to homogeneity. Biochemical studies of nusA protein reveal that it inhibits in vitro RNA chain elongation by E. coli RNA polymerase with a variety of templates. Two modes of inhibition are found. Inhibition of elongation with poly[d(A-T] ) template is completely competitive with nucleoside triphosphates and shows an inhibitory constant (Ki) of 3 X 10(-7) M. In contrast, inhibition of elongation with T7 DNA as template is mixed. One component of the inhibition is competitive with nucleoside triphosphate substrates and is reversed at elevated substrate concentrations. A second inhibitory component remains even at saturating substrate concentrations; this sequence-dependent mode of inhibition shows a much lower Ki of 2 X 10(-8) M. The existence of two different modes of inhibition might be explained if two molecules of nusA protein can bind to each RNA polymerase complex. The interaction of nusA protein with elongating RNA polymerase molecules is not processive but appears to be characterized by rapid association and dissociation. Under proper conditions, a sigma-nusA cycle [Greenblatt, J., & Li, J. (1981) Cell (Cambridge, Mass.) 24, 421-428] can be demonstrated in vitro in which each polymerase goes through multiple rounds of transcription involving successive interactions with sigma and the nusA protein.

Bacterial Proteins↗

PubMed1984