The carboxyl-terminal repeat domain of RNA polymerase II is not required for transcription factor Sp1 to function in vitro.

We show that the mammalian transcription Sp1 stimulates accurate transcription in a partially fractionated RNA polymerase II-dependent system from Drosophila cultured cells. Moreover, the extent of stimulation is equal for intact RNA polymerase II (polymerase IIA) and polymerase lacking the unique carboxyl-terminal domain of the largest subunit (polymerase IIB). We conclude that in this system Sp1 interacts with a component of the transcription machinery, other than the carboxyl-terminal domain, which is preserved between mammals and insects.

Analysis of the gene encoding the largest subunit of RNA polymerase II in Drosophila.

We have characterized RpII215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster. DNA sequencing and nuclease S1 analyses provided the primary structure of this gene, its 7 kb RNA and 215 kDa protein products. The amino-terminal 80% of the subunit harbors regions with strong homology to the beta' subunit of Escherichia coli RNA polymerase and to the largest subunits of other eukaryotic RNA polymerases. The carboxyl-terminal 20% of the subunit is composed of multiple repeats of a seven amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The homology domains, as well as the unique carboxyl-terminal structure, are considered in the light of current knowledge of RNA polymerase II and the properties of its largest subunit. Additionally, germline transformation demonstrated that a 9.4 kb genomic DNA segment containing the alpha-amanitin-resistant allele, RpII215C4, includes all sequences required to produce amanitin-resistant transformants.

The C-terminal repeat domain of RNA polymerase II largest subunit is essential in vivo but is not required for accurate transcription initiation in vitro.

DNA sequence analysis of RpII215, the gene that encodes the Mr215,000 subunit of RNA polymerase II (EC 2.7.7.6) in Drosophila melanogaster, reveals that the 3'-terminal exon includes a region encoding a C-terminal domain composed of 42 repeats of a seven-residue amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A hemi- and homozygous lethal P-element insertion into the coding sequence of this domain causes premature translation termination and therefore truncation of the protein, leaving only 20 heptamer repeats. While loss of approximately 50% of the repeat structure in this mutant is a lethal event in vivo, enzyme containing the truncated subunit remains capable of accurate initiation at promoters in vitro. Moreover, treatment of purified intact RNA polymerase II with protease, to remove the entire repeat domain, does not eliminate the enzyme's ability to initiate accurately in vitro. Possible in vivo functions for this unusual protein domain are considered in light of these results.

Germ-line transformation involving DNA from the period locus in Drosophila melanogaster: overlapping genomic fragments that restore circadian and ultradian rhythmicity to per0 and per- mutants.

P-element-mediated transformations involving DNA fragments from the period (per) clock gene of Drosophila melanogaster have shown that several subsegments of the locus restore rhythmicity to per0 or per- mutants. Such fragments overlap in a genomic region complementary to one transcript, a 4.5-kb RNA which is probably the per message, in that it is necessary and (in terms of expression from this X-chromosomal locus) sufficient for the fly's circadian rhythms. It is also at least necessary for the high-frequency oscillations normally produced by courting males as they vibrate their wings. The entirety of the 4.5-kb transcript is not necessary for rather strong rhythmicity; nor does it seem to be sufficient, in transformants, for wild-type behavioral phenotypes. A 0.9-kb RNA, homologous to genomic region immediately adjacent to the source of the 4.5-kb species, oscillates in its abundance over the course of a day; but coverage of this transcript source in several transformants carrying a per0 mutation--which eliminates the 0.9-kb RNA's oscillation--does not restore rhythmicity. All of the independently isolated arrhythmic mutations tested were covered by the same array of overlapping per+-derived DNA fragments, implying that the only portion of the locus which has mutated to arrhythmicity is complementary to the 4.5-kb transcript.

P-element transformation with period locus DNA restores rhythmicity to mutant, arrhythmic Drosophila melanogaster.

Mutations at the period (per) locus of Drosophila melanogaster disrupt several biological rhythms. Molecular cloning of DNA sequences encompassing the per+ locus has allowed germ-line transformation experiments to be carried out. Certain subsegments of the per region, transduced into the genome of arrhythmic pero flies, restore rhythmicity in circadian locomotor behavior and the male's courtship song.

Molecular analysis of the period locus in Drosophila melanogaster and identification of a transcript involved in biological rhythms.

We have isolated and analyzed DNA sequences encompassing the period (per) locus of Drosophila melanogaster. The location of this clock gene was delimited by the molecular mapping of chromosome aberrations at or very near the per locus. At least five RNAs are transcribed from this region. One of these transcripts, a 0.9 kb species, is strongly implicated in per's control of biological rhythms. Two independently isolated arrhythmic mutations at the per locus dramatically reduce the level of this transcript. Furthermore, the level of the 0.9 kb transcript is strongly modulated during a light/dark cycle. We discuss evidence, from previously reported genetic and phenotypic analysis of per's function, suggesting that this region may be complex and that several gene products from the per region, including this 0.9 kb transcript, may be involved in the different aspects of normal rhythmicity influenced by this clock gene.

Purification and characterization of coliphage N4 RNA polymerase II activity from infected cell extracts.

The soluble components of the RNA polymerase activity (N4 RNA polymerase II) required for coliphage N4 middle RNA synthesis have been purified to homogeneity using a complementation assay described elsewhere. These soluble components are found to exhibit the properties of a DNA-dependent RNA polymerase which is resistant to both rifampicin and streptolydigin and transcribes denatured N4 DNA with marked preference but with little selectivity for the middle region of the N4 genome. In its native form, the activity is composed of one Mr = 40,000 polypeptide (p4, the product of N4 cistron 4) and one Mr = 30,000 polypeptide (p7, the product of N4 cistron 3). Its physical properties and the mechanism of transcription selectivity are discussed.

Bacteriophage N4-induced transcribing activities in E. coli. III. A third cistron required for N4 RNA polymerase II activity.

The requirement of a third gene product of molecular weight 15,000 for coliphage N4 middle RNA synthesis is described. Therefore, three proteins of molecular weights 15,000, 30,000, and 40,000, which constitute the N4 RNA polymerase II activity, are required for N4 middle RNA synthesis. A complementation assay for N4 RNA polymerase II activity, which requires a DNA-membrane complex carrying the 15,000-MW protein and a supernatant providing the 30,000- and 40,000-MW proteins, has been developed. This assay, which reproduces the requirement for the cistron 5 gene product in vitro provides a means for the purification of the soluble components of the N4 RNA polymerase II activity (to be reported elsewhere). The cistron 5 gene product is also required for N4 RNA polymerase II to synthesize RNAs utilizing middle promoters resident in recombinant plasmids. Mechanisms by which the cistron 5 gene product affects N4 RNA polymerase II transcription in vivo and in vitro are discussed.