agr-Amanitin Tolerance in Mycophagous Drosophila.

Six species of Drosophila were tested for tolerance to the mushroom toxin alpha-amanitin, a potent inhibitor of RNA polymerase II. Three nonmycophagous species-D. melanogaster, D. immigrans, and D. pseudoobscura-showed very low survival and long development times in the presence of amanitin. Three mycophagous species-D. putrida, D. recens, and D. tripunctata-showed little or no sensitivity. Analysis in vitro indicated that this tolerance is not based on alteration of the molecular structure of RNA polymerase II.

Positive patches and negative noodles: linking RNA processing to transcription?

A speculative model is presented that proposes specific mechanisms for effecting co-transcriptional splice site selection in pre-mRNAs. The model envisions that certain splicing factors containing arginine-rich, positively charged regions bind via these positive patches to the hyperphosphorylated, negatively charged tail of elongating RNA polymerase II. Thus tethered to the transcription machinery, these splicing factors gain access to signals in nascent transcripts as they emerge from the polymerase.

Dynamic interaction between a Drosophila transcription factor and RNA polymerase II.

We have purified factor 5, a Drosophila RNA polymerase II transcription factor. Factor 5 was found to be required for accurate initiation of transcription from specific promoters and also had a dramatic effect on the elongation properties of RNA polymerase II. Kinetic studies suggested that factor 5 stimulates the elongation rate of RNA polymerase II on a dC-tailed, double-stranded template by reducing the time spent at the numerous pause sites encountered by the polymerase. The factor was found to be composed of two polypeptides (34 and 86 kilodaltons). Both subunits bound tightly to pure RNA polymerase II but were not bound to polymerase in elongation complexes. Our results suggest that factor 5 interacts briefly with the paused polymerase molecules and catalyzes a conformational change in them such that they adopt an elongation-competent conformation.

Sites of P element insertion and structures of P element deletions in the 5' region of Drosophila melanogaster RpII215.

Several P element insertion and deletion mutations near the 5' end of Drosophila melanogaster RpII215 have been examined by nucleotide sequencing. Two different sites of P element insertion, approximately 90 nucleotides apart, have been detected in this region of the gene. Therefore, including an additional site of P element insertion within the coding region, there are at least three distinct sites of P element insertion at RpII215. Both 5' sites are within a noncoding portion of transcribed sequences. The sequences of four revertants of one P element insertion mutation (D50) indicate that the P element is either precisely deleted or internally deleted to restore RpII215 activity. Partial internal deletions of the P element result in different RpII215 activity levels, which appear to depend on the specific sequences that remain after excision.

The yeast carboxyl-terminal repeat domain kinase CTDK-I is a divergent cyclin-cyclin-dependent kinase complex.

Saccharomyces cerevisiae CTDK-I is a protein kinase complex that specifically and efficiently hyperphosphorylates the carboxyl-terminal repeat domain (CTD) of RNA polymerase II and is composed of three subunits of 58, 38, and 32 kDa. The kinase is essential in vivo for normal phosphorylation of the CTD and for normal growth and differentiation. We have now cloned the genes for the two smaller kinase subunits, CTK2 and CTK3, and found that they form a unique, divergent cyclin-cyclin-dependent kinase complex with the previously characterized largest subunit protein CTK1, a cyclin-dependent kinase homolog. The CTK2 gene encodes a cyclin-related protein with limited homology to cyclin C, while CTK3 shows no similarity to other known proteins. Copurification of the three gene products with each other and CTDK-I activity by means of conventional chromatography and antibody affinity columns has verified their participation in the complex in vitro. In addition, null mutations of each of the genes and all combinations thereof conferred very similar growth-impaired, cold-sensitive phenotypes, consistent with their involvement in the same function in vivo. These characterizations and the availability of all of the genes encoding CTDK-I and reagents derivable from them will facilitate investigations into CTD phosphorylation and its functional consequences both in vivo and in vitro.

Analyses of promoter-proximal pausing by RNA polymerase II on the hsp70 heat shock gene promoter in a Drosophila nuclear extract.

Analyses of Drosophila cells have revealed that RNA polymerase II is paused in a region 20 to 40 nucleotides downstream from the transcription start site of the hsp70 heat shock gene when the gene is not transcriptionally active. We have developed a cell-free system that reconstitutes this promoter-proximal pausing. The paused polymerase has been detected by monitoring the hyperreactivity of thymines in the transcription bubble toward potassium permanganate. The pattern of permanganate reactivity for the hsp70 promoter in the reconstituted system matches the pattern found on the promoter after it has been introduced back into files by P-element-mediated transposition. Matching patterns of permanganate reactivity are also observed for a non-heat shock promoter, the histone H3 promoter. Further analysis of the hsp70 promoter in the reconstituted system reveals that pausing does not depend on sequence-specific interactions located immediately downstream from the pause site. Sequences upstream from the TATA box influence the recruitment of polymerase rather than the efficiency of pausing. Kinetic analysis indicates that the polymerase rapidly enters the paused state and remains stably in this state for at least 25 min. Further analysis shows that the paused polymerase will initially resume elongation when Sarkosyl is added but loses this capacity within minutes of pausing. Using an alpha-amanitin-resistant polymerase, we provide evidence that promoter-proximal pausing does not require the carboxy-terminal domain of the polymerase.

Mapping mutations in genes encoding the two large subunits of Drosophila RNA polymerase II defines domains essential for basic transcription functions and for proper expression of developmental genes.

We have mapped a number of mutations at the DNA sequence level in genes encoding the largest (RpII215) and second-largest (RpII140) subunits of Drosophila melanogaster RNA polymerase II. Using polymerase chain reaction (PCR) amplification and single-strand conformation polymorphism (SSCP) analysis, we detected 12 mutations from 14 mutant alleles (86%) as mobility shifts in nondenaturing gel electrophoresis, thus localizing the mutations to the corresponding PCR fragments of about 350 bp. We then determined the mutations at the DNA sequence level by directly subcloning the PCR fragments and sequencing them. The five mapped RpII140 mutations clustered in a C-terminal portion of the second-largest subunit, indicating the functional importance of this region of the subunit. The RpII215 mutations were distributed more broadly, although six of eight clustered in a central region of the subunit. One notable mutation that we localized to this region was the alpha-amanitin-resistant mutation RpII215C4, which also affects RNA chain elongation in vitro. RpII215C4 mapped to a position near the sites of corresponding mutations in mouse and in Caenorhabditis elegans genes, reinforcing the idea that this region is involved in amatoxin binding and transcript elongation. We also mapped mutations in both RpII215 and RpII140 that cause a developmental defect known as the Ubx effect. The clustering of these mutations in each gene suggests that they define functional domains in each subunit whose alteration induces the mutant phenotype.

Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II.

The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription.

The C-terminal domain (CTD) of the large subunit of RNA polymerase II plays a role in transcription and RNA processing. Yeast ESS1, a peptidyl-prolyl cis/trans isomerase, is involved in RNA processing and can associate with the CTD. Using several types of assays we could not find any evidence of an effect of Pin1, the human homolog of ESS1, on transcription by RNA polymerase II in vitro or on the expression of a reporter gene in vivo. However, an inhibitor of Pin1, 5-hydroxy-1,4-naphthoquinone (juglone), blocked transcription by RNA polymerase II. Unlike N-ethylmaleimide, which inhibited all phases of transcription by RNA polymerase II, juglone disrupted the formation of functional preinitiation complexes by modifying sulfhydryl groups but did not have any significant effect on either initiation or elongation. Both RNA polymerases I and III, but not T7 RNA polymerase, were inhibited by juglone. The primary target of juglone has not been unambiguously identified, although a site on the polymerase itself is suggested by inhibition of RNA polymerase II during factor-independent transcription of single-stranded DNA. Because of its unique inhibitory properties juglone should prove useful in studying transcription in vitro.

Functional studies of the carboxy-terminal repeat domain of Drosophila RNA polymerase II in vivo.

To understand the in vivo function of the unique and conserved carboxy-terminal repeat domain (CTD) of RNA polymerase II largest subunit (RpII215), we have studied RNA polymerase II biosynthesis, activity and genetic function in Drosophila RpII215 mutants that possessed all (C4), half (W81) or none (IIt) of the CTD repeats. We have discovered that steady-state mRNA levels from transgenes encoding a fully truncated, CTD-less subunit (IIt) are essentially equal to wild-type levels, whereas the levels of the CTD-less subunit itself and the amount of polymerase harboring it (Pol IIT) are significantly lower than wild type. In contrast, for the half-CTD mutant (W81), steady-state mRNA levels are somewhat lower than for wild type or IIt, while W81 subunit and polymerase amounts are much less than wild type. Finally, we have tested genetically the ability of CTD mutants to complement (rescue) partially functional RpII215 alleles and have found that IIt fails to complement whereas W81 complements partially to completely. These results suggest that removal of the entire CTD renders polymerase completely defective in vivo, whereas eliminating half of the CTD results in a polymerase with significant in vivo activity.

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.

Frequent Imprecise Excision among Reversions of a P Element-Caused Lethal Mutation in Drosophila.

RpII215(D) (50) (= D50) is a lethal mutation caused by the insertion of a 1.3-kb P element 5' to sequences encoding the largest (215 kilodaltons) subunit of Drosophila RNA polymerase II. In dysgenic males D50 reverted to nonlethality at frequencies ranging from 2.6 to 6.5%. These reversions resulted from loss of P element sequences. Genetic tests of function and restriction enzyme analysis of revertant DNAs revealed that 35% or more of the reversion events were imprecise excisions. Two meiotic mutations that perturb excision repair and postreplication repair (mei-9(a) and mei-41(D5), respectively) had no influence on reversion frequency but may have increased the proportion of imprecise excisions. We suggest that these excisions are by-products of, rather than intermediates in, the transposition process.

Co-transcriptional splicing of pre-messenger RNAs: considerations for the mechanism of alternative splicing.

Nascent transcripts are the true substrates for many splicing events in mammalian cells. In this review we discuss transcription, splicing, and alternative splicing in the context of co-transcriptional processing of pre-mRNA. The realization that splicing occurs co-transcriptionally requires two important considerations: First, the cis-acting elements in the splicing substrate are synthesized at different times in a 5' to 3' direction. This dynamic view of the substrate implies that in a 100 kb intron the 5' splice site will be synthesized as much as an hour before the 3' splice site. Second, the transcription machinery and the splicing machinery, which are both complex and very large, are working in close proximity to each other. It is therefore likely that these two macromolecular machines interact, and recent data supporting this notion is discussed. We propose a model for co-transcriptional pre-mRNA processing that incorporates the concepts of splice site-tethering and dynamic exon definition. Also, we present a dynamic view of the alternative splicing of FGF-R2 and suggest that this view could be generally applicable to many regulated splicing events.

An activity necessary for in vitro transcription is a DNase inhibitor.

A phosphocellulose flowthrough fraction required for accurate transcription in vitro by RNA polymerase II was found to contain a DNase inhibitor which was necessary to maintain template integrity (Price D.H., Sluder A.E. & Greenleaf A.L. (1987) J. Biol. Chem. 262, 3244-3255). Starting with a Drosophila Kc cell nuclear extract, the DNase inhibitory activity has been purified 19,000-fold. In combination with the other necessary fractions, the highly purified inhibitor continues to support reconstruction of transcription. It thus appears to be the only required activity in the original phosphocellulose flowthrough fraction. The inhibitor is a protein which does not bind to DNA or inhibit DNase I, so that it has also been useful in assays for DNA binding proteins in crude, DNase-contaminated fractions.

Identifying a transcription factor interaction site on RNA polymerase II.

We have generated a series of fusion proteins carrying portions of subunit IIc, the second largest subunit of Drosophila RNA polymerase I, and have used them in a domain interference assay to identify a fragment of the IIc subunit that carries the binding site for a basal transcription factor. Fusion proteins carrying a subunit IIc fragment spanning residues Ala519-Gly992 strongly inhibit promoter-driven transcription in both unfractionated nuclear extracts and in reconstituted systems. The same fusion proteins similarly inhibit dTFIIF stimulation of Pol II elongation on dC-tailed templates, suggesting that the IIc(A519-G992) fragment, which carries conserved regions D-H, interferes with transcription by binding to dTFIIF. Finally, dTFIIF can be specifically cross-linked to a GST-IIc(A519-G992) fusion protein or to subunit IIc in intact Pol II.

CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae.

We previously purified a yeast protein kinase that specifically hyperphosphorylates the carboxyl-terminal repeat domain (CTD) of RNA polymerase II largest subunit and showed that this CTD kinase consists of three subunits of 58, 38, and 32 kDa. We have now cloned, sequenced, and characterized CTK1, the gene encoding the 58 kDa alpha subunit. The CTK1 gene product contains a central domain homologous to catalytic subunits of other protein kinases, notably yeast CDC28, suggesting that the 58 kDa subunit is catalytic. Cells that carry a disrupted version of the CTK1 gene lack the characterized CTD kinase activity, grow slowly and are cold-sensitive, demonstrating that the CTK1 gene product is essential for CTD kinase activity and normal growth. While ctk1 mutant cells do contain phosphorylated forms of the RNA polymerase II largest subunit, these forms differ from those found in wild type cells, implicating CTK1 as a component of the physiologically significant CTD phosphorylating machinery. As befitting an enzyme with a nuclear function, the N-terminal region of the CTK1 protein contains a nuclear targeting signal.

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.

Modulation of RNA polymerase II elongation efficiency by C-terminal heptapeptide repeat domain kinase I.

Hyperphosphorylation of the C-terminal heptapeptide repeat domain (CTD) of the RNA polymerase II largest subunit has been suggested to play a key role in regulating transcription initiation and elongation. To facilitate investigating functional consequences of CTD phosphorylation we developed new templates, the double G-less cassettes, which make it possible to assay simultaneously the level of initiation and the efficiency of elongation. Using these templates, we examined the effects of yeast CTD kinase I or CTD kinase inhibitors on transcription and CTD phosphorylation in HeLa nuclear extracts. Our results showed that polymerase II elongation efficiency and CTD phosphorylation are greatly reduced by CTD kinase inhibitors, whereas both are greatly increased by CTD kinase I; in contrast, transcription initiation is much less affected. These results demonstrate that CTD kinase I modulates the elongation efficiency of RNA polymerase II and are consistent with the idea that one function of CTD phosphorylation is to promote effective production of long transcripts by stimulating the elongation efficiency of RNA polymerase II.

A mutation in the largest subunit of RNA polymerase II alters RNA chain elongation in vitro.

An in vitro transcription system which utilized a semisynthetic DNA template (Kadesch, T. R., and Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295) was developed and used to compare RNA chain elongation by wild type and mutant RNA polymerases II of Drosophila. With this template, all of the active polymerases rapidly initiated RNA chains at synthetic single-stranded sites at the ends of the DNA, and then entered a long (15 to 20 min) period of elongation through duplex regions of template before any measurable termination occurred. A comparison of wild type and mutant polymerase activities during this elongation phase indicated that a mutation to amanitin resistance reduces the rate at which the enzyme elongates transcripts. The reduced elongation rate of the mutant was associated with an altered substrate Km. Because the polymerase II mutation is in the largest enzyme subunit (Greenleaf, A. L. (1983) J. Biol. Chem. 258, 13403-13406), these results demonstrate a functional role for this subunit during RNA chain elongation.