Bacteriophage promoters P R and P RM direct RNA synthesis in divergent orientations from start sites 82 base pairs apart. We had previously determined that the presence on the same DNA fragment of a wild type P R promoter interfered with the utilization of the P RM promoter. The results reported here concern the effects of changing the distance between the start sites by insertion or deletion of 5 or 10 base pairs. Three different techniques (run-off transcription, gel mobility shift, and permanganate probing) were employed to monitor complex formation at P RM . Unexpectedly we find that deletion of 10 base pairs between the start sites abolishes the interference, whereas insertion of 10 base pairs does not. Deletion of 5 base pairs, however, essentially prevents joint complex formation at P R and P RM . These findings suggest several ways in which for the wild type separation of the two promoters the utilization of P RM could be affected by an RNA polymerase at P R . In addition to direct steric interference, these include the obstruction of access to DNA sites necessary for optimal contact with the RNA polymerase.Promoters P R and P RM of bacteriophage direct the synthesis of nonoverlapping, divergent transcripts originating from start sites separated by 82 base pairs. We (1, 2) as well as Gussin and co-workers (3, 4) have shown that the presence of the P R promoter has a negative effect on the ability of RNA polymerase to form open complexes at P RM . The use of P R mutants allowed Gussin's group to demonstrate that conversely, the presence of an RNA polymerase at P RM negatively affects the utilization of a (weakened) P R promoter as well (5).Open complex formation at the wild type P R promoter occurs within seconds (6, 7), but formation of the complex at P RM is several orders of magnitude slower, on the time scale of tens of minutes (1,3,8). Thus nearly every RNA polymerase binding at P RM does so in the context of another polymerase situated at P R . Our own work (1, 2) as well as more extensive kinetic analysis by Gussin and his group (3, 4) has demonstrated that the interference with complex formation at P RM manifests itself not at the initial bimolecular (binding) step but rather in some subsequent step involving conformational transitions in RNA polymerase and/or DNA. The interference slows down but does not prevent open complex formation at P RM ; ultimately open complexes at both promoters do co-exist on the same DNA fragment (Refs. 1 and 9 and this work).Reduction of the distance between P R and P RM by one base pair further slows open complex formation at P RM (4). The effects of considerably shorter separation between the P R and P RM promoters can be assessed from studies on other lambdoid phages. For the 434 phage where the distance between the start sites of P R and P RM is about 65 base pairs, the Ϫ35 regions of the two promoters overlap and concurrent binding of RNA polymerases at the two promoters is not observed (10). For P22 the interpromoter distance is even shorter, making it quite unl...
Although thyroid hormone has been known for many years to be a potent regulator of skeletal maturation in vivo, it has not definitively been determined whether this effect is a result of a direct or indirect action of the hormone. Previous in vivo studies have suggested that thyroid hormone may stimulate longitudinal bone growth by increasing the secretion of growth hormone; however, growth hormone alone is unable to stimulate cartilage maturation. There are also indications that thyroid hormone is able to act directly on growth plate chondrocytes through growth hormone-independent mechanisms. In this study, we demonstrate that rat growth plate chondrocytes in vivo express genes encoding three of the four isoforms of the thyroid hormone receptors described to date, but the corresponding protein can only be detected for the TR␣1 and TR1 isoforms of the receptor. As has been noted in other tissues, there is generally poor correlation between the mRNA levels for each isoform and the relative amount of corresponding protein as measured by immunoblotting, suggesting the possibility that receptor expression may be regulated by post-transcriptional mechanisms. (J Bone Miner Res 1999;14:1550-1556)
Thyroid hormone has been known for over 50 years to be a potent regulator of skeletal maturation at the growth plate. The receptor for thyroid hormone has been discovered to be a member of the nuclear hormone receptor superfamily. Retinoic acid and 1,25(OH)? vitamin D3. whose receptors also belong to this nuclear hormone receptor family, have been implicated in the control of chondrocyte proliferation and differentiation at the growth plate. Recent studies demonstrate that the receptors for thyroid hormone, retinoic acid, and vitamin D bind to a similar DNA response element in the promoter region of target genes and may form heterodimers to regulate gene transcription in target cells. These observations led us to hypothesize that the retinoic acid andlor vitamin D signaling pathways may interact with thyroid hormone signaling at the molecular level to modulate growth plate chondrocyte differentiation. Using a chemically defined, serum-free model of growth plate chondrocyte maturation, both all-mim retinoic acid and 1 ,?5(0H): vitamin D3 markedly inhibited thyroid hormone-induced terminal differentiation in a dose-dependent manner. In the absence of thyroid hormone, retinoic acid stimulated alkaline phosphatase activity modestly at the highest dose used. however neither retinoic acid nor 1,95(0H)2 vitamin D3 induced expression of type X collagen mRNA. We conclude that retinoic acid and vitamin D are likely to be antagonists of thyroid hormone signaling in the growth plate. C) 9001 Orthopaedic Reserach Society. Published by
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