Nuclear factor 1 (NF1) affects accurate termination and multiple-round transcription by human RNA polymerase III.
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We have shown previously that the TFIIIC1/TFIIIC1' fraction interacts specifically with the VA1 terminator regions to affect both termination and initiation/reinitiation of transcription by human RNA polymerase III. Here, we further purified the VA1 terminator-binding factor to apparent homogeneity and found, by peptide sequence analysis, that it belongs to the NF1 protein family. NF1 interacts specifically with the NF1-binding sites within the terminator regions of the VA1 gene and with two subunits (TFIIIC220 and TFIIIC110) of human TFIIIC2. Immunodepletion with anti-NF1 antibodies dramatically decreases transcription from the VA1 template in nuclear extract, and mutation at the NF1-binding site in the terminator region of the VA1 gene selectively affects multiple-round transcription (reinitiation of transcription) and termination. In addition, NF1 acts in conjunction with TFIIIC to promote accurate termination by RNA polymerase III on a C-tailed VA1 template. (+info)
Nonsense-mediated decay of mutant waxy mRNA in rice.
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Two rice (Oryza sativa) waxy mutations of the Japonica background were shown to contain approximately 20% of the fully spliced mRNA relative to the wild type. Sequencing analysis of the entire waxy genes of the two mutants revealed the presence of premature translation termination codons in exon 2 and exon 7. These results indicated that the lower accumulation of fully spliced RNA in the mutants was caused by nonsense-mediated decay (NMD), which is an RNA surveillance system universally found in eukaryotes. It is interesting that levels of RNA retaining intron 1 were not changed by premature nonsense codons, suggesting that splicing may be linked with NMD in plants, as previously found in mammalian cells. Measurements of the half-lives of waxy RNAs in transfected rice protoplasts indicated that the half-life of waxy RNA with a premature nonsense codon was 3.3 times shorter than that without a premature nonsense codon. Because the wild-type waxy transcripts, which are derived from the Wx(b) gene predominantly distributed among Japonica rice, have been shown to be less efficiently spliced and their alternative splicing has been documented, we examined whether these splicing properties influenced the efficiency of NMD. However, no effects were observed. These results established that NMD occurs in rice waxy RNA containing a premature nonsense codon. (+info)
Acetylation of TAF(I)68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription.
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Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF-I. In exploring the functional significance of the promoter-proximal terminator, we found that TTF-I associates with the p300/CBP-associated factor PCAF, suggesting that TTF-I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAF(I)68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAF(I)68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF-IB/SL1 being required for rDNA transcription, the NAD(+)-dependent histone deacetylase mSir2a deacetylates TAF(I)68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF-IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals. (+info)
The viral RNA polymerase H4L subunit is required for Vaccinia virus early gene transcription termination.
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Vaccinia virus early gene transcription is catalyzed by a multisubunit virion form of RNA polymerase that possesses a unique subunit, H4L. Prior studies from this laboratory showed that the NH(2)-terminal domain of H4L, containing amino acids 1-195, interacts with the COOH-terminal end of nucleoside triphosphate phosphohydrolase I (NPH I), an ATPase that is employed in early gene transcription termination. Carboxyl-terminal deletion mutations of NPH I lose both the ability to mediate transcription termination and binding to H4L, providing evidence that the interaction between NPH I and H4L is required for termination. In order to test this model further, antibodies raised against segments of H4L were tested for their ability to inhibit transcription termination in vitro. A bead-bound template was employed in these studies, which permitted us to separate transcription initiation from elongation and termination. Antibodies raised against H4L amino acids 1-256 inhibited termination in an in vitro assay using virus-infected cell extracts lacking NPH I, but antibodies raised against H4L amino acids 568-795 did not. Preincubation of anti-H4L(1-256) antibodies with H4L fragments 1-256 or 1-195 prevented antibody inhibition of termination, demonstrating that inhibition was mediated by antibody binding to one or more epitopes in the NH(2)-terminal end of H4L. Antibody inhibition of termination is reduced in wild type virus-infected cell extracts containing NPH I. Furthermore, preincubation of a NPH I minus cell extract with NPH I prior to antibody addition, or readdition of NPH I to isolated ternary complexes prepared in the absence of NPH I, prevented antibody inhibition of transcription termination. These data show that NPH I and the inhibitory antibodies compete for a binding site(s) on H4L, providing further evidence that the H4L subunit of the vaccinia virus RNA polymerase plays a direct role in transcription termination. (+info)
Development of an inducible pol III transcription system essentially requiring a mutated form of the TATA-binding protein.
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We attempted to devise a transcription system in which a particular DNA sequence of interest could be inducibly expressed under the control of a modified polymerase III (pol III) promoter. Its activation requires a mutated transcription factor not contained endogenously in human cells. We constructed such a promoter by fusing elements of the beta-lactamase gene of Escherichia coli, containing a modified TATA-box and a pol III terminator, to the initiation region of the human U6 gene. This construct functionally resembles a 5'-regulated pol III gene and its transcribed segment can be exchanged for an arbitrary sequence. Its transcription in vitro by pol III requires the same factors as the U6 gene with the major exception that the modified TATA-box of this construct only interacts with a TATA-binding protein (TBP) mutant (TBP-DR2) but not with TBP wild-type (TBPwt). Its transcription therefore requires TBP-DR2 exclusively instead of TBPWT: In order to render the system inducible, we fused the gene coding for TBP-DR2 to a tetracycline control element and stably transfected this new construct into HeLa cells. Induction of such a stable and viable clone with tetracycline resulted in the expression of functional TBP-DR2. This system may conceptually be used in the future to inducibly express an arbitrary DNA sequence in vivo under the control of the above mentioned promoter. (+info)
Balanced branching in transcription termination.
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The theory of stochastic transcription termination based on free-energy competition [von Hippel, P. H. & Yager, T. D. (1992) Science 255, 809-812 and von Hippel, P. H. & Yager, T. D. (1991) Proc. Natl. Acad. Sci. USA 88, 2307-2311] requires two or more reaction rates to be delicately balanced over a wide range of physical conditions. A large body of work on glasses and large molecules suggests that this balancing should be impossible in such a large system in the absence of a new organizing principle of matter. We review the experimental literature of termination and find no evidence for such a principle, but do find many troubling inconsistencies, most notably, anomalous memory effects. These effects suggest that termination has a deterministic component and may conceivably not be stochastic at all. We find that a key experiment by Wilson and von Hippel [Wilson, K. S. & von Hippel, P. H. (1994) J. Mol. Biol. 244, 36-51] thought to demonstrate stochastic termination was an incorrectly analyzed regulatory effect of Mg(2+) binding. (+info)
Identity elements in tRNA-mediated transcription antitermination: implication of tRNA D- and T-arms in mRNA recognition.
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tRNA-mediated transcription antitermination has been shown to control the expression of several amino acid biosynthesis operons and aminoacyl-tRNA-synthetase-encoding genes in Gram-positive bacteria. A model originally put forward by Grundy & Henkin describes the conserved structural features of the leader sequences of these operons and genes. Two sequences of 3 and 4 nt, respectively, take a central position in this model and are thought to be responsible for the binding of the system-specific uncharged tRNA, an interaction which would stabilize the antiterminator conformation of the leader. Here a further evolution of this model is presented based on an analysis of trp regulation in Lactococcus lactis in which a function is assigned to hitherto unexplained conserved structures in the leader sequence. It is postulated that the mRNA-tRNA interaction involves various parts of the tRNA in addition to the anticodon and the acceptor in the original model and that these additional interactions contribute to the recognition of a specific tRNA, and hence to the specificity and efficacy of the regulatory response. (+info)
Molecular characterization of Pseudomonas putida KT2440 rpoH gene regulation.
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The rpoH gene of Pseudomonas putida KT2440 encoding the heat-shock sigma factor sigma(32) was cloned and sequenced, and the translated gene product was predicted to be a protein of 32.5 kDa. The unambiguous role of the gene as a sigma factor was confirmed because the cloned P. putida gene complemented the growth defect, at 37 and 42 degrees C, of an Escherichia coli rpoH mutant strain. Primer extension analysis showed that in P. putida the rpoH gene is expressed from three promoters in cells growing at 30 degrees C. Two of them, P1 and P3, share homology with the sigma(70)-dependent promoters, while the third one, P2, shows a typical sigma(24)-consensus sequence. The pattern of transcription initiation of the rpoH gene did not change in response to different stresses, i.e. a sudden heat shock or the addition of aromatic compounds. However, the predicted secondary structure of the 5' region of the mRNA derived from the three different promoters suggests regulation at the level of translation efficiency and/or mRNA half-life. An inverted repeat sequence located 20 bp downstream of the rpoH stop codon was shown to function as a terminator in vivo in P. putida growing at temperatures from 18 to 42 degrees C. (+info)