The form of RNA polymerase II (RNAPII) engaged in transcriptional elongation was isolated. Elongating RNAPII was associated with a novel multisubunit complex, termed elongator, whose stable interaction was dependent on a hyperphosphorylated state of the RNAPII carboxy-terminal domain (CTD). A free form of elongator was also isolated, demonstrating the discrete nature of the complex, and free elongator could bind directly to RNAPII. The gene encoding the largest subunit of elongator, ELP1, was cloned. Phenotypes of yeast elp1 delta cells demonstrated an involvement of elongator in transcriptional elongation as well as activation in vivo. Our data indicate that the transition from transcriptional initiation to elongation involves an exchange of the multiprotein mediator complex for elongator in a reaction coupled to CTD hyperphosphorylation.

The elongator complex is a major component of the RNA polymerase II (RNAPII) holoenzyme responsible for transcriptional elongation in yeast. Here we identify Elp3, the 60-kilodalton subunit of elongator/RNAPII holoenzyme, as a highly conserved histone acetyltransferase (HAT) capable of acetylating core histones in vitro. In vivo, ELP3 gene deletion confers typical elp phenotypes such as slow growth adaptation, slow gene activation, and temperature sensitivity. These results suggest a role for a novel, tightly RNAPII-associated HAT in transcription of DNA packaged in chromatin.

Eukaryotic cells use multiple, highly conserved mechanisms to contend with ultraviolet-light-induced DNA damage. One important response mechanism is transcription-coupled repair (TCR), during which DNA lesions in the transcribed strand of an active gene are repaired much faster than in the genome overall. In mammalian cells, defective TCR gives rise to the severe human disorder Cockayne's syndrome (CS). The best-studied CS gene, CSB, codes for a Swi/Snf-like DNA-dependent ATPase, whose yeast homologue is called Rad26 (ref. 4). Here we identify a yeast protein, termed Def1, which forms a complex with Rad26 in chromatin. The phenotypes of cells lacking DEF1 are consistent with a role for this factor in the DNA damage response, but Def1 is not required for TCR. Rather, def1 cells are compromised for transcript elongation, and are unable to degrade RNA polymerase II (RNAPII) in response to DNA damage. Our data suggest that RNAPII stalled at a DNA lesion triggers a coordinated rescue mechanism that requires the Rad26-Def1 complex, and that Def1 enables ubiquitination and proteolysis of RNAPII when the lesion cannot be rapidly removed by Rad26-promoted DNA repair.

Free and elongating (DNA-bound) forms of RNA polymerase II were separated from yeast. Most cellular polymerase II was found in the elongating fraction, which contained all enzyme phosphorylated on the C-terminal domain and none of the 15-subunit mediator of transcriptional regulation. These and other findings suggest that mediator enters and leaves initiation complexes during every round of transcription, in a process that may be coupled to C-terminal domain phosphorylation.

A novel yeast gene, ELP2, is shown to encode the 90-kDa subunit of the Elongator complex and elongating RNA polymerase II holoenzyme. ELP2 encodes a protein with eight WD40 repeats, and cells lacking the gene display typical elp phenotypes, such as temperature and salt sensitivity. Generally, different combinations of double and triple ELP gene deletions cause the same phenotypes as single ELP1, ELP2, or ELP3 deletion, providing genetic evidence that the ELP gene products work together in a complex.The Elongator complex is a major component of native RNA polymerase II (RNAPII) 1 elongation complexes and co-purifies with elongating RNAPII holoenzyme after its extraction from ternary DNA/RNA/RNAPII complexes (1). Elongator interacts directly with RNAPII and stable association is dependent on the C-terminal domain of the largest polymerase subunit being in a hyperphosphorylated state. Another RNAPII-associated complex, Mediator (2, 3), is exclusively associated with nonphosphorylated RNAPII and is recycled at transcriptional initiation (4). It is therefore an attractive model that Elongator displaces Mediator at the initiation/elongation transition in response to transcription factor IIH-mediated C-terminal domain phosphorylation and thereafter assists RNAPII during elongation (1). Elongator comprises three subunits of 150, 90, and 60 kDa. The gene encoding p150, ELP1/IKI3, appears to have no significant homology to protein domains of known function (1). It does, however, have a well conserved Schizosaccharomyces pombe homologue of unknown function and, furthermore, might be the yeast counterpart of human IKAP protein (5). The gene encoding p60, ELP3, is highly conserved, with likely functional homologues found in many higher eukaryotes, such as Caenorhabditis elegans, Drosophila melanogaster, and man (6).2 Elp3 is a histone acetyltransferase, pointing to a role for Elongator in chromatin disruption during transcript elongation. Deletion of ELP1 or ELP3 confers a peculiar slow start phenotype, manifested as a pronounced delay in adaptation to new growth conditions. Elongator mutants are also salt-and temperature-sensitive. We have found that these phenotypes are in all likelihood a result of delays in the activation of genes required for growing under the new conditions. For example, activation of the genes GAL1-10, PHO5, and ENA1, which are normally repressed and turned on 50 -1000-fold by appropriate growth conditions, is delayed and reduced in the mutants (1, 6).Here we show that the 90-kDa subunit of Elongator is encoded by the ELP2 gene, which encodes a protein with eight WD40 repeats. ELP2 deletion, and, indeed, all combinations of ELP deletions, confer phenotypes similar to those previously described for ELP1 and ELP3. EXPERIMENTAL PROCEDURES Expression and Purification of Recombinant Elp2-The open reading frame encoding Elp2 was amplified by the polymerase chain reaction from yeast genomic DNA, using primers that introduced a hexahistidine tag and a unique BamHI site at the end encoding the N terminus, and ...

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Scatter factor (SF), a glycoprotein produced by cultured fibroblasts, acts in vitro on epithelial cells causing separation and increased local motility. In this study, the polypeptide was purified to apparent homogeneity in high yields with conserved biological activity from medium conditioned by ras-transformed NIH 3T3 cells, by a three-step procedure involving ammonium sulphate fractionation, cation-exchange and hydroxyapatite chromatography. After purification, SF specific activity increased from approximately 0.3 units/microgram in unprocessed conditioned medium to approximately 5 units/ng, and cumulative recovery of biological activity was approximately 38%. Treatment of pure SF with N-glycanase resulted in a decreased Mr, but no concomitant effect was observed on biological activity. Proteolytic activity was absent from samples of both partially purified and pure SF. Our biochemical studies showed that SF, which is highly aggregated in low-ionic-strength media, is not aggregated in 0.4 M-salt. Under non-reducing conditions, pure SF migrated as a single stained band at Mr 67,000 on SDS/PAGE, and biological activity was eluted from unstained gels with an identical Mr. SF was electrofocused sharply at pI 8.5 with no degradation of activity. From ultracentrifugation studies (under non-aggregating conditions), the sedimentation coefficient of active SF was 3.7 S and f.p.l.c. molecular sieve chromatography indicated a Stokes' radius of 2.95 nm. The calculated Mr from these data was 61,400. The appearance of three stained polypeptides of Mr 82,000, 57,000 and 32,000 derived from the Mr-67,000 constituent after reduction with mercaptoethanol suggests that SF may be a heterodimer of Mr-57,000 and -32,000 subunits. Data from protein sequence analysis of the hydroxyapatite-purified protein confirms that SF has sequence identity with both rat hepatocyte growth factor and human fibroblast tumour cytotoxic factor.

Although the crystal structure of nucleosome core particle is essentially symmetric in the vicinity of the dyad the linker histone binds asymmetrically in this region to select one single high affinity site from potentially two equivalent sites. To try to resolve this apparent paradox we mapped to bp resolution the dyads and rotational settings of nucleosomc core particles reassembled on synthetic tandemly repeating 20 bp D N A sequences. We observed, in agreement with previous observations (Travers and Klug, Phil.Trans. Roy. SOC. London, B317537-561,1987; Hayes et al., Proc. Natl. Acad. Sci. USA, 877405-7409, 1990) that the helical repeat on each side of the dyad cluster is 10 bp maintaining register with the sequence repeat and second, that this register changes by 2 bp in the vicinity of the dyad. The additional 2 bp required to effect the change in the rotational settings is accommodated by an asymmetric adjustment immediately adjacent to the dyad. We suggest that this asymmetry in the helical repeat is sequencedependent and could direct the binding of the linker histone to a single preferred site. The implications of such binding will be discussed in the context of mechanisms for the establishment and maintenance of transcriptional repression.Gene regulation involves the generation of a local chromatin topology that is conducive to transcription. Several classes of chromatin remodelling activity have been shown to play a role in this process. ATP dependent chromatin remodelling activities use energy derived from the hydrolysis of ATP to alter the structure of chromatin making it more accessible for transcription factor binding. The yeast SWI/SNF complex is the founding member of this family of ATP-dependent chromatin remodelling activities.We have developed a model system that we have used to study the ability of the SWI/SNF complex to alter chromatin structure. Using this system, we find that the SWI/SNF is able to the position of nucleosomes along DNA. To investigate the mechanism by which these complexes function in more detail we have developed new assays for chromatin remodelling. We have used these to study the remodelling of nucleosomes assembled onto D N A fragments derived from the MMTV LlX region. The results of this work suggest that the yeast SWI/SNF complex can alter chromatin structure in ways that do not just involve changes to nucleosome positioning.Transcription of eukaryotic genes is regulated on several levels. Chromatin domains harbouring potentially active genes need to be remodelled to allow transcription factors access; these factors need to be recruited to the promoter; and finally, the processes of actual transcriptional initiation and elongation are also subject to control. Our studies are mostly focused on transcriptional elongation. We have isolated the form of RNA polymerase 11 which is responsible for this process and shown it to be fundamentally different from the form involved in initiation. Our work has thus shed light on the basic mechanisms governing the transition from tr...