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J M Rientjes

Publications and source records attributed to J M Rientjes.

8 recordsLinked to original sources
PubMed1998

Different positioning of the ligand-binding domain helix 12 and the F domain of the estrogen receptor accounts for functional differences between agonists and antagonists.

The estrogen receptor is capable of binding a diverse set of ligands that are broadly categorized as agonists or antagonists, depending on their abilities to induce or interfere with transcriptional responsiveness. We show, using a fusion protein assay for ligand-binding which does not rely on transcriptional responsiveness, that agonists and antagonists differently position the C-terminus of the ligand-binding domain (helix 12) and the F domain. Upon antagonist binding, the F domain interferes with the fusion protein activity. Mutational disruption of helix 12 alters the position of the F domain, imposing interference after agonist or antagonist binding. Genetically selected inversion mutations where only agonists, but not antagonists, induce interference are similarly reliant on helix 12 and F domain positioning. Our results demonstrate that agonists and antagonists differently position helix 12 and implicate the F domain in mechanisms of antagonist action.

Amino Acid Sequence↗
PubMed1997

FLP recombinase/estrogen receptor fusion proteins require the receptor D domain for responsiveness to antagonists, but not agonists.

The ligand-binding domains of steroid receptors convey ligand-dependent regulation to certain proteins to which they are fused. Here we characterize fusion proteins between a site-specific recombinase, FLP, and steroid receptor ligand-binding domains. These proteins convert ligand binding into DNA recombination. Thus, ligand binding is directly coupled to an enzyme activity that is easily measured by DNA rearrangements or heritable genetic changes in marker gene expression, as opposed to the multiple events leading to transcription. Recombination by a FLP-estrogen receptor (FLP-EBD) fusion is activated by all tested estrogens, whether agonists or antagonists, indicating that all induce EBD release from the 90-kDa heat shock protein complex. Altering the distance between FLP and the EBD domain in the fusion proteins, by reducing the included length of the estrogen receptor D domain, affects ligand efficacy. A FLP-EBD with no D domain shows reduced inducibility by agonists and, unexpectedly, complete insensitivity to induction by all antagonists tested. A FLP-EBD including some D domain shows a ligand-inducible phenotype intermediate to those displayed by FLP-EBDs containing all or none of the D domain. Thus, we observed a tethered interference between FLP and the EBD domains that differs depending on the distance between the two domains, the conformations induced by agonists or antagonists, and which presents a previously undetectable distinction between estrogen agonists and antagonists in yeast.

Amino Acid Sequence↗
PubMed1995

Evolutionarily conserved structural elements are critical for processing of Internal Transcribed Spacer 2 from Saccharomyces cerevisiae precursor ribosomal RNA.

Structural features of Internal Transcribed Spacer 2 (ITS2) important for the correct and efficient removal of this spacer from Saccharomyces cerevisiae pre-rRNA were identified by in vivo mutational analysis based upon phylogenetic comparison with its counterparts from four different yeast species. Compatibility between ITS2 structure and the S. cerevisiae processing machinery was found to have been maintained over only a short evolutionary distance, in contrast to the situation for ITS1. Nevertheless, cis-acting elements required for correct and efficient processing are confined predominantly to those regions of the spacer that show the highest degree of evolutionary conservation. Mutation or deletion of each of these regions severely reduced production of mature 26 S, but not 17 S rRNA, mainly by impeding processing of the 29 SB precursor. In some cases, however, conversion of 29SA into 29 SB pre-rRNA also appeared to be affected. Deletion of non-conserved segments, on the other hand, caused little or no disturbance in processing. Surprisingly, some combinations of such individually neutral deletions had a severe negative effect on the removal of ITS2, suggesting a requirement for a higher-order structure of ITS2. Finally, even structural alterations of ITS2 that did not noticeably affect processing, significantly reduced the growth rate of cells that exclusively express the mutant rDNA units. We take this as further evidence for a direct role of ITS2 in the formation of fully functional 60 S ribosomal subunits.

Base Sequence↗
PubMed1994

Separate structural elements within internal transcribed spacer 1 of Saccharomyces cerevisiae precursor ribosomal RNA direct the formation of 17S and 26S rRNA.

Structural features of Internal Transcribed Spacer 1 (ITS1) that direct its removal from Saccharomyces cerevisiae pre-rRNA during processing were identified by an initial phylogenetic approach followed by in vivo mutational analysis of specific structural elements. We found that S. cerevisiae ITS1 can functionally be replaced by the corresponding regions from the yeasts Torulaspora delbrueckii, Kluyveromyces lactis and Hansenula wingei, indicating that structural elements required in cis for processing are evolutionarily conserved. Despite large differences in size, all ITS1 regions conform to the secondary structure proposed by Yeh et al. [Biochemistry 29 (1990) 5911-5918], showing five domains (I-V; 5'-->3') of which three harbour an evolutionarily highly conserved element. Removal of most of domain II, including its highly conserved element, did not affect processing. In contrast, highly conserved nucleotides directly downstream of processing site A2 in domain III play a major role in production of 17S, but not 26S rRNA. Domain IV and V are dispensable for 17S rRNA formation although an alternative, albeit inefficient, processing route to mature 17S rRNA may be mediated by a conserved region in domain IV. Each of these two domains is individually sufficient for efficient production of 26S rRNA, suggesting two independent processing pathways. We conclude that ITS1 is organized into two functionally and structurally distinct halves.

Base Sequence↗
PubMed1993

Transcriptional regulation of the gene encoding cholesterol 7 alpha-hydroxylase in the rat.

The cytochrome P450 enzyme, cholesterol 7 alpha-hydroxylase (CYP7A), catalyses the first and rate-limiting step in the conversion of cholesterol to bile acids. Expression of the CYP7A gene is under complex physiological control, encompassing amongst others a feedback down-regulation by bile acids. Using the CYP7A cDNA of the rat as a probe, we isolated a rat genomic clone containing the 5' part of the gene, including approximately 3.6 kb of upstream sequences. Sequence analysis revealed the presence of several putative regulatory elements. Transient expression analyses of transfected primary hepatocytes demonstrated that the major transcription-activating region is located in the proximal 145 nucleotide (nt). Upon addition of taurocholate to the culture, a significant reduction of the transcriptional activity was observed, suggesting the presence of a bile acid-responsive element in the proximal region of the CYP7A promoter. In addition, evidence was obtained for the presence of a thyroxine-responsive site further upstream. After addition of taurocholate, steady-state CYP7A mRNA levels, as judged by Northern analysis of hepatocyte RNA, are eightfold reduced. On the other hand, the transcriptional activity of CYP7A, as shown both in CAT assays and run-on experiments, revealed only a threefold decrease. These experiments suggest that both transcriptional control and regulation of CYP7A mRNA stability play an important part in the feedback regulation of CYP7A activity in the rat.

Animals↗
PubMed1991

rRNA binding domain of yeast ribosomal protein L25. Identification of its borders and a key leucine residue.

We have delineated the region of yeast ribosomal protein L25 responsible for its specific binding to 26 S rRNA by a novel approach using in vitro synthesized, [35S]methionine-labeled fragments as well as point mutants of the L25 protein. The rRNA binding capacity of these mutant polypeptides was tested by incubation with an in vitro transcribed, biotinylated fragment of yeast 26 S rRNA that contains the complete L25 binding site. Protein-rRNA interaction was assayed by binding of the rRNA-r-protein complex to streptavidin-agarose followed either by analysis of the bound polypeptide by SDS/polyacrylamide gel electrophoresis or by precipitation with trichloroacetic acid. Our results show that the structural elements necessary and sufficient for specific interaction of L25 with 26 S rRNA are contained in the region bordered by amino acids 62 and 126. The remaining parts of the protein, in particular the C-terminal 16 residues, while not essential for binding, do enhance its affinity for 26 S rRNA. To test whether, as suggested by the results of the deletion experiments, the evolutionarily conserved sequence motif K120KAYVRL126 is involved in rRNA binding, we replaced the leucine residue at position 126 by either isoleucine or lysine. The first substitution did not affect binding. The second, however, completely abolished the specific rRNA binding capacity of the protein. Thus, Leu126, and possibly the whole conserved sequence motif, plays a key role in binding of L25 to 26 S rRNA.

Base Sequence↗
PubMedDate not supplied

Processing of eukaryotic pre-rRNA: the role of the transcribed spacers.

The 17-18S, 5.8S, and 25-28S rRNA species of eukaryotic cells are produced by a series of nucleolytic reactions that liberate the mature rRNAs from the large primary precursor transcript synthesized by RNA polymerase 1. Whereas the order of the cleavage reactions has long been established, until recently little information was available on their molecular details, such as the nature of the proteins, including the nucleolytic enzymes, involved and the signals directing the processing machinery to the correct sites. This situation is now rapidly changing, in particular where yeast is concerned. The use of recently developed systems for in vivo mutational analysis of yeast rDNA has considerably enhanced our knowledge of cis-acting structural features within the pre-rRNA, in particular the transcribed spacer sequences, that are critical for correct and efficient removal of these spacers. The same systems also allow a link to be forged between trans-acting processing factors and these cis-acting elements. In this paper, we will focus predominantly on the nature and role of the cis-acting processing elements as identified in the transcribed spacer regions of Saccharomyces cerevisiae pre-rRNA.

Base Sequence↗