Biomedical subjects

J Saez-Vasquez

Publications and source records attributed to J Saez-Vasquez.

4 recordsLinked to original sources

PubMed2001

RNA polymerase I holoenzyme-promoter complexes include an associated CK2-like protein kinase.

In eukaryotes, RNA polymerase I (pol I) transcribes the tandemly repeated genes that encode the precursor of 18S, 5.8S and 25S ribosomal RNAs. In plants and animals, the pol I enzyme can be purified in a holoenzyme form that is self-sufficient for promoter binding and accurate, promoter-dependent transcription in a cell-free system. In this report, we show that a casein kinase 2 (CK2)-like protein kinase co-purifies with pol I holoenzyme activity purified from broccoli (Brassica oleracea). Using an immobilized template assay, we show that the CK2-like activity is part of the protein-DNA complex that results upon binding of the holoenzyme to the rRNA gene promoter. The CK2 activity phosphorylates a similar set of holoenzyme proteins both before and after promoter binding. These data provide further evidence that pol I holoenzyme activity can be attributed to a single, multi-protein complex self-sufficient for promoter association and accurate, promoter-dependent transcription.

Brassica↗

PubMed2000

RNA polymerase I holoenzyme-promoter interactions.

In plants and animals, RNA polymerase I (pol I) can be purified in a form that is self-sufficient for accurate rRNA gene promoter-dependent transcription and that has biochemical properties suggestive of a single complex, or holoenzyme. In this study, we examined the promoter binding properties of a highly purified Brassica pol I holoenzyme activity. DNase I footprinting revealed protection of the core promoter region from approximately -30 to +20, in good agreement with the boundaries of the minimal promoter defined by deletion analyses (-33 to +6). Using conventional polyacrylamide electrophoretic mobility shift assays (EMSA), protein-DNA complexes were mostly excluded from the gel. However, agarose EMSA revealed promoter-specific binding activity that co-purified with promoter-dependent transcription activity. Titration, time-course, and competition experiments revealed the formation or dissociation of a single protein-DNA complex. This protein-DNA complex could be labeled by incorporation of radioactive ribonucleotides into RNA in the presence of alpha-amanitin, suggesting that the polymerase I enzyme is part of the complex. Collectively, these results suggest that transcriptionally competent pol I holoenzymes can associate with rRNA gene promoters in a single DNA binding event.

Brassica↗

PubMed1999

RNA polymerase I transcription in a Brassica interspecific hybrid and its progenitors: Tests of transcription factor involvement in nucleolar dominance.

In interspecific hybrids or allopolyploids, often one parental set of ribosomal RNA genes is transcribed and the other is silent, an epigenetic phenomenon known as nucleolar dominance. Silencing is enforced by cytosine methylation and histone deacetylation, but the initial discrimination mechanism is unknown. One hypothesis is that a species-specific transcription factor is inactivated, thereby silencing one set of rRNA genes. Another is that dominant rRNA genes have higher binding affinities for limiting transcription factors. A third suggests that selective methylation of underdominant rRNA genes blocks transcription factor binding. We tested these hypotheses using Brassica napus (canola), an allotetraploid derived from B. rapa and B. oleracea in which only B. rapa rRNA genes are transcribed. B. oleracea and B. rapa rRNA genes were active when transfected into protoplasts of the other species, which argues against the species-specific transcription factor model. B. oleracea and B. rapa rRNA genes also competed equally for the pol I transcription machinery in vitro and in vivo. Cytosine methylation had no effect on rRNA gene transcription in vitro, which suggests that transcription factor binding was unimpaired. These data are inconsistent with the prevailing models and point to discrimination mechanisms that are likely to act at a chromosomal level.

Binding, Competitive↗

PubMed1997

Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription in vitro.

RNA polymerase I (pol I) is a nuclear enzyme whose function is to transcribe the duplicated genes encoding the precursor of the three largest ribosomal RNAs. We report a cell-free system from broccoli (Brassica oleracea) inflorescence that supports promoter-dependent RNA pol I transcription in vitro. The transcription system was purified extensively by DEAE-Sepharose, Biorex 70, Sephacryl S300, and Mono Q chromatography. Activities required for pre-rRNA transcription copurified with the polymerase on all four columns, suggesting their association as a complex. Purified fractions programmed transcription initiation from the in vivo start site and utilized the same core promoter sequences required in vivo. The complex was not dissociated in 800 mM KCl and had a molecular mass of nearly 2 MDa based on gel filtration chromatography. The most highly purified fractions contain approximately 30 polypeptides, two of which were identified immunologically as RNA polymerase subunits. These data suggest that the occurrence of a holoenzyme complex is probably not unique to the pol II system but may be a general feature of eukaryotic nuclear polymerases.

Arabidopsis↗