Reverse Genetics of Drosophila RNA Polymerase II: Identification and Characterization of Rpii140, the Genomic Locus for the Second-Largest Subunit

We have used a reverse genetics approach to isolate genes encoding two subunits of Drosophila melanogaster RNA polymerase II. RpII18 encodes the 18-kDa subunit and maps cytogenetically to polytene band region 83A. RpII140 encodes the 140-kDa subunit and maps to polytene band region 88A10:B1,2. Focusing on RpII140, we used in situ hybridization to map this gene to a small subinterval defined by the endpoints of a series of deficiencies impinging on the 88A/B region and showed that it does not represent a previously known genetic locus. Two recently defined complementation groups, A5 and Z6, reside in the same subinterval and thus were candidates for the RpII140 locus. Phenotypes of A5 mutants suggested that they affect RNA polymerase II, in that the lethal phase and the interaction with developmental loci such as Ubx resemble those of mutants in the gene for the largest subunit, RpII215. Indeed, we have achieved complete genetic rescue of representative recessive lethal mutations of A5 with a P-element construct containing a 9.1-kb genomic DNA fragment carrying RpII140. Interestingly, the initial construct also rescued lethal alleles in the neighboring complementation group, Z6, revealing that the 9.1-kb insert carries two genes. Deleting coding region sequences of RpII140, however, yielded a transformation vector that failed to rescue A5 alleles but continued to rescue Z6 alleles. These results strongly support the conclusion that the A5 complementation group is equivalent to the genomic RpII140 locus.     

Mechanisms of Mutagenesis by a Bulky DNA Lesion at the Guanine N7 Position

The RpII215 locus encodes the large subunit of RNA polymerase II (polII). Three of 22 RpII215 alleles cause a synergistic enhancement of the mutant phenotype elicited by mutations in the Ultrabithorax (Ubx) locus. We have recovered and analyzed three new mutations that suppress this enhancement. All three mutations map to the RpII215 locus. In addition to suppressing the Ubx enhancement of other RpII215 alleles, two of the new mutations, JH1 and WJK2, themselves enhance Ubx. RpII215 alleles can be placed into three classes based on their ability to enhance Ubx. Class I alleles, including Ubl, C4, C11, JH1, and WJK2, enhance Ubx when heterozygous with class II alleles, which include wild-type RpII215. Class III alleles, which include amorphic alleles, do not enhance Ubx. The third new mutation, WJK1, is a conditional amorphic allele, which behaves like a class III allele at 29 degrees but like a class II allele at 19 degrees. Another mutant phenotype is caused by certain RpII215 alleles, including all class I alleles. This phenotype is a synergistic enhancement of a mutant phenotype elicited by mutations at the Delta (Dl) locus. Unlike the enhancement of Ubx, the enhancement of Dl is not dependent upon antagonistic interactions between different classes of RpII215 alleles.     

Synonymous rates at the RpII215 gene of Drosophila: variation among species and across the coding region

The region encompassing the RpII215 gene that encodes the largest component of the RNA polymerase II complex (1889 amino acids) has been sequenced in Drosophila subobscura, D. madeirensis, D. guanche, and D. pseudoobscura. Nonsynonymous divergence estimates (Ka) indicate that this gene has a very low rate of amino acid replacements. Given its low Ka and constitutive expression, synonymous substitution rates are, however, unexpectedly high. Sequence comparisons have allowed the molecular clock hypothesis to be tested. D. guanche is an insular species and it is therefore expected to have a reduced effective size relative to D. subobscura. The significantly higher rate of synonymous substitutions detected in the D. guanche lineage could be explained if synonymous mutations behave as nearly neutral. Significant departure from the molecular clock hypothesis for synonymous and nonsynonymous substitutions was detected when comparing the D. subobscura, D. pseudoobscura, and D. melanogaster lineages. Codon bias and synonymous divergence between D. subobscura and D. melanogaster were negatively correlated across the RpII215 coding region, which indicates that selection coefficients for synonymous mutations vary across the gene. The C-terminal domain (CTD) of the RpII215 protein is structurally and functionally differentiated from the rest of the protein. Synonymous substitution rates were significantly different in both regions, which strongly indicates that synonymous mutations in the CTD and in the non-CTD regions are under detectably different selection coefficients.     

Amanitin-resistant RNA polymerase II mutations are in the enzyme's largest subunit

A fragment of the Drosophila melanogaster RpIIC4 locus, which encodes the RNA polymerase II subunit that determines amanitin sensitivity, was inserted into a bacterial plasmid cloning vehicle useful for over-production of hybrid proteins. Two plasmid constructions encoded hybrid proteins that reacted with antibodies against D. melanogaster RNA polymerase II. Use of subunit-specific antibodies indicated that these hybrid proteins displayed antigenic determinants unique to the largest polypeptide (215 kDa) of the enzyme. This RpII locus, the site at which mutations to amanitin-resistance occur, must therefore encode the largest polymerase II subunit.     

The two steps of poly(A)-dependent termination, pausing and release, can be uncoupled by truncation of the RNA polymerase II carboxyl-terminal repeat domain

The carboxyl-terminal repeat domain (CTD) of RNA polymerase II is thought to help coordinate events during RNA metabolism. The mammalian CTD consists of 52 imperfectly repeated heptads followed by 10 additional residues at the C terminus. The CTD is required for cleavage and polyadenylation in vitro. We studied poly(A)-dependent termination in vivo using CTD truncation mutants. Poly(A)-dependent termination occurs in two steps, pause and release. We found that the CTD is required for release, the first 25 heptads being sufficient. Neither the final 10 amino acids nor the variant heptads of the second half of the CTD were required. No part of the CTD was required for poly(A)-dependent pausing--the poly(A) signal could communicate directly with the body of the polymerase. By removing the CTD, pausing could be observed without being obscured by release. Poly(A)-dependent pausing appeared to operate by slowing down the polymerase, such as by down-regulation of a positive elongation factor. Although the first 25 heptads supported undiminished poly(A)-dependent termination, they did not efficiently support events near the promoter involved in abortive elongation. However, the second half of the CTD, including the final 10 amino acids, was sufficient for these functions.     

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

Summary 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 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 -amanitinresistant allele, RpII215 ^C4 , includes all sequences required to produce amanitin-resistant transformants.     

Genetic and molecular variation in the RpII215 region of Drosophila melanogaster

Summary The RpII215 region of the X chromosome of Drosophila melanogaster was investigated to identify genetic functions and correlate these with the known molecular organization of the region. Five genetic loci were identified in a subregion that is reported to transcribe nine or more messages. One locus is nod, which causes meiotic abnormalities, and three other loci are recessive lethal mutations whose developmental lesions are unknown. The fifth and most mutable of the loci is RpII215, which encodes the 215,000 dalton subunit of RNA polymerase II. Mutant effects of RpII215 alleles include: temperature-dependent (heat and cold) survival, altered sensitivity to -amanitin, male sterility, maternal effects and epistatic enhancement of mutant effects of other loci.