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274 records · Page 16Linked to original sources
PubMed1989

Multiple mutations of the first gene of a dimeric tRNA gene abolish in vitro tRNA gene transcription.

Eukaryotic tRNA expression initiates with transcription by RNA polymerase III and requires two additional protein factors and two regions within the tRNA gene (the 5'-internal control region (ICR) or A-box and the 3'-ICR or B-box). Using a reconstituted Saccharomyces cerevisiae RNA polymerase III system, the transcription of various 5'-ICR, 3'-ICR, and double mutation alleles of the Schizosaccharomyces pombe sup3-e dimeric tRNA gene were studied. The sup3-e tRNA locus consists of an upstream serine tRNA gene and a downstream initiator methionine tRNA gene which are transcribed as a dimeric precursor and processed to give two tRNAs. Only the ICRs of the tRNA(Ser) gene are active in directing dimeric gene transcription. Mutations in the 3'-ICR of the tRNA(Ser) gene reduce transcription of the dimer more than those in the 5'-ICR. Mutations in the 5'-ICR were found which greatly increased or decreased transcription of the dimer, while base changes in the 3'-ICR were only found to decrease transcription. This suggests a modulatory role for the 5'-ICR in transcription regulation. Mutation of the methionine tRNA gene ICR has little effect on sup3-e transcription, and no detectable transcripts initiate from the methionine tRNA gene when the tRNA(Ser) gene promoter is inactivated by mutation. Comparison with transcription studies of other mutant tRNA genes suggests that nucleotides sites within the ICRs, such as nucleotides 8, 10, 13, 18, and 19 in the 5'-ICR and 48, 53, 56, 57, and 58 in the 3'-ICR, appear to have evolved universal importance for RNA polymerase III transcription in eukaryotes. Thus these ICR sequences may play a critical role in regulation of tRNA expression.

Cloning, Molecular↗
PubMed1989

Purification and characterization of transcription factor IIIC2.

Transcription factor IIIC2 (TFIIIC2), together with other transcription factors (TFIIIB and TFIIIC1), is required for the in vitro transcription of tRNA and adenovirus VA genes by RNA polymerase III. Previous studies have shown that TFIIIC2 is a high molecular weight (approximately 500,000) protein which binds with high affinity to the B-box promoter element of tRNA-type genes. A polypeptide of Mr approximately 250,000 is in close association with DNA in the specific complex between TFIIIC2 and the B-box promoter element. Here we describe the purification of TFIIIC2 by a factor of approximately 25,000 from nuclear extracts of HeLa cells by ionic exchange, affinity, and hydrophobic chromatography and sedimentation velocity centrifugation. The most purified fractions contain polypeptides of approximately 230 kDa (corresponding to the polypeptide which can be cross-linked to VA1 DNA), 110, 100, 80, and 60 kDa which co-sediment with TFIIIC2 B-box specific binding and in vitro transcriptional activities.

Adenoviruses, Human↗
PubMed1987

DNA-binding properties and characterization of human transcription factor TFIIIC2.

Interaction between the B-block region of adenovirus VA1 DNA and the human RNA polymerase III transcription factor (TFIII) C2 was analyzed using a gel DNA-binding assay. The retarded band corresponding to the specific complex between TFIIIC2 and the regulatory B-block region was identified by DNase I footprint analysis, competition experiments, and gel shift assays using mutated and truncated virus-associated (VA) 1 DNA probes. The equilibrium constants for the binding reaction with the complete VA1 gene were determined. TFIIIC2 was found to bind to non-specific DNA sequences with a relatively low affinity (equilibrium constant Kn = 6 x 10(4) M-1), and to the B-block sequence with a high affinity (specific constant Ks = 2 x 10(11) M-1). Assuming one site per molecule, the total concentration of binding sites [C0] in the TFIIIC2-containing fractions ranged between 0.6 and 1.6 x 10(-10) M. This corresponded to 1500 TFIIIC2 molecules extracted per 293 cell. Sedimentation analysis of TFIIIC2 on a sucrose gradient showed both VA1 DNA binding activity and in vitro transcription activity cosedimenting with an apparent coefficient of 17-18 S, consistent with a molecular weight of 400-500 kDa. UV cross-linking of a 5-bromo 2'-deoxyuridine-containing, 32P-labeled VA1 probe with the TFIIIC2 fraction, followed by DNase I digestion and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed a single 32P-labeled band migrating as a 250-kDa polypeptide. Compared with the sedimentation data, this result suggests that native TFIIIC2 may be a dimer of the approximately 250-kDa polypeptide.

Adenoviruses, Human↗
PubMed1997

Differential kinetics of transcription complex assembly distinguish oocyte and somatic 5S RNA genes of Xenopus.

Differential transcription of the Xenopus gene families encoding the oocyte and somatic 5S ribosomal RNAs can be reproduced in vitro with cell-free extracts prepared from Xenopus oocytes and unfertilized eggs. The transcriptional activities of these genes as assayed in these in vitro systems are a consequence of large differences in the rates of assembly of active transcription complexes. The somatic 5S genes sequester limiting transcription factors much more rapidly than the corresponding oocyte 5S genes and, as a consequence, are far more active. However, once transcription complexes are formed, these complexes are stable on both of these genes. Previous studies have established that transcription factors IIIA and IIIC are sufficient to form a stable protein-DNA complex on the somatic 5S gene. The rate of formation of the stable TFIIIA+C complex for the oocyte gene is far slower than that for the somatic 5S gene. Insertion of the DNA binding site for TFIIIC2 (the B-block promoter element from tRNA genes) into the 3' flanking region of a synthetic oocyte 5S gene increases the transcription efficiency and rate of transcription complex assembly of this gene relative to the parent gene lacking the B-block element. Our results support a model in which competition for limiting transcription factors plays a pivotal role in establishing differential transcription of the two classes of 5S genes during early embryogenesis.

Animals↗