TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines

We have previously identified a cellular protein kinase activity termed TAK that specifically associates with the HIV types 1 and 2 Tat proteins. TAK hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II in vitro in a manner believed to activate transcription [Herrmann, C. H. & Rice, A. P. (1995) J. Virol. 69, 1612–1620]. We show here that the catalytic subunit of TAK is a known human kinase previously named PITALRE, which is a member of the cyclin-dependent family of proteins. We also show that TAK activity is elevated upon activation of peripheral blood mononuclear cells and peripheral blood lymphocytes and upon differentiation of U1 and U937 promonocytic cell lines to macrophages. Therefore, in HIV-infected individuals TAK may be induced in T cells following activation and in macrophages following differentiation, thus contributing to high levels of viral transcription and the escape from latency of transcriptionally silent proviruses.

An essential component of a C-terminal domain phosphatase that interacts with transcription factor IIF in Saccharomyces cerevisiae

One of the essential components of a phosphatase that specifically dephosphorylates the Saccharomyces cerevisiae RNA polymerase II (RPII) large subunit C-terminal domain (CTD) is a novel polypeptide encoded by an essential gene termed FCP1. The Fcp1 protein is localized to the nucleus, and it binds the largest subunit of the yeast general transcription factor IIF (Tfg1). In vitro, transcription factor IIF stimulates phosphatase activity in the presence of Fcp1 and a second complementing fraction. Two distinct regions of Fcp1 are capable of binding to Tfg1, but the C-terminal Tfg1 binding domain is dispensable for activity in vivo and in vitro. Sequence comparison reveals that residues 173–357 of Fcp1 correspond to an amino acid motif present in proteins of unknown function predicted in many organisms.

Ultrastructural Analysis of Transcription and Splicing in the Cell Nucleus after Bromo-UTP Microinjection

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.

The Drosophila melanogaster Homologue of the Xeroderma Pigmentosum D Gene Product Is Located in Euchromatic Regions and Has a Dynamic Response to UV Light-induced Lesions in Polytene Chromosomes

The XPD/ERCC2/Rad3 gene is required for excision repair of UV-damaged DNA and is an important component of nucleotide excision repair. Mutations in the XPD gene generate the cancer-prone syndrome, xeroderma pigmentosum, Cockayne’s syndrome, and trichothiodystrophy. XPD has a 5′- to 3′-helicase activity and is a component of the TFIIH transcription factor, which is essential for RNA polymerase II elongation. We present here the characterization of the Drosophila melanogaster XPD gene (DmXPD). DmXPD encodes a product that is highly related to its human homologue. The DmXPD protein is ubiquitous during development. In embryos at the syncytial blastoderm stage, DmXPD is cytoplasmic. At the onset of transcription in somatic cells and during gastrulation in germ cells, DmXPD moves to the nuclei. Distribution analysis in polytene chromosomes shows that DmXPD is highly concentrated in the interbands, especially in the highly transcribed regions known as puffs. UV-light irradiation of third-instar larvae induces an increase in the signal intensity and in the number of sites where the DmXPD protein is located in polytene chromosomes, indicating that the DmXPD protein is recruited intensively in the chromosomes as a response to DNA damage. This is the first time that the response to DNA damage by UV-light irradiation can be visualized directly on the chromosomes using one of the TFIIH components.

Assembly of Lampbrush Chromosomes from Sperm Chromatin

We have examined the behavior of demembranated sperm heads when injected into the germinal vesicle (GV) of amphibian oocytes. Xenopus sperm heads injected into Xenopus GVs swelled immediately and within hours began to stain with an antibody against RNA polymerase II (Pol II). Over time each sperm head became a loose mass of chromosome-like threads, which by 24–48 h resolved into individually recognizable lampbrush chromosomes (LBCs). Although LBCs derived from sperm are unreplicated single chromatids, their morphology and immunofluorescent staining properties were strikingly similar to those of the endogenous lampbrush bivalents. They displayed typical transcriptionally active loops extending from an axis of condensed chromomeres, as well as locus-specific “landmarks.” Experiments with [3H]GTP and actinomycin D demonstrated that transcription was not necessary for the initial swelling of the sperm heads and acquisition of Pol II but was required for maintenance of the lampbrush loops. Splicing was not required at any stage during formation of sperm LBCs. When Xenopus sperm heads were injected into GVs of the newt Notophthalmus, the resulting sperm LBCs displayed very long loops with pronounced Pol II axes, like those of the endogenous newt LBCs; as expected, they stained with antibodies against newt-specific proteins. Other heterologous injections, including sperm heads of the frog Rana pipiens and the zebrafish Danio rerio in Xenopus GVs, confirm that LBCs can be derived from taxonomically distant organisms. The GV system should help identify both cis- and trans-acting factors needed to convert condensed chromatin into transcriptionally active LBCs. It may also be useful in producing cytologically analyzable chromosomes from organisms whose oocytes do not go through a typical lampbrush phase or cannot be manipulated by current techniques.

Coiled Bodies and U2 snRNA Genes Adjacent to Coiled Bodies Are Enriched in Factors Required for snRNA Transcription

A significant percentage of the gene clusters that contain the human genes for U1 small nuclear RNA (snRNA) or for U2 snRNA have been found associated with small nuclear domains, known as coiled bodies. We show here, by immunofluorescent labeling of human cells, that coiled bodies are enriched in factors required for the transcription of these snRNA genes. The 45-kDa γ-subunit of the transcription factor, proximal element sequence-binding transcription factor (PTF), which is specific for the snRNA genes, was found in high concentrations in coiled bodies, along with the general transcription factor TATA-box binding protein and a subset of RNA polymerase II. We show that the transcription factors and RNA polymerase II are concentrated in irregularly shaped domains that not only overlap with coiled bodies but also extend to their immediate surroundings. Fluorescent in situ hybridization showed that these domains can overlap with U2 snRNA genes adjacent to coiled bodies. In addition, we found the domains to contain newly synthesized RNA, visualized by 5-bromo-uridine triphosphate labeling. Our data suggest that coiled bodies are involved in the expression of snRNA genes, which leads us to propose the model that coiled bodies are associated with snRNA genes to facilitate and regulate their transcription. These findings point to a general principle of higher order organization of gene expression in the nucleus.

Human and Yeast Cdk-activating Kinases (CAKs) Display Distinct Substrate Specificities

Cell cycle progression is controlled by the sequential functions of cyclin-dependent kinases (cdks). Cdk activation requires phosphorylation of a key residue (on sites equivalent to Thr-160 in human cdk2) carried out by the cdk-activating kinase (CAK). Human CAK has been identified as a p40MO15/cyclin H/MAT1 complex that also functions as part of transcription factor IIH (TFIIH) where it phosphorylates multiple transcriptional components including the C-terminal domain (CTD) of the large subunit of RNA polymerase II. In contrast, CAK from budding yeast consists of a single polypeptide (Cak1p), is not a component of TFIIH, and lacks CTD kinase activity. Here we report that Cak1p and p40MO15 have strikingly different substrate specificities. Cak1p preferentially phosphorylated monomeric cdks, whereas p40MO15 preferentially phosphorylated cdk/cyclin complexes. Furthermore, p40MO15 only phosphorylated cdk6 bound to cyclin D3, whereas Cak1p recognized monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3. We also found that cdk inhibitors, including p21CIP1, p27KIP1, p57KIP2, p16INK4a, and p18INK4c, could block phosphorylation by p40MO15 but not phosphorylation by Cak1p. Our results demonstrate that although both Cak1p and p40MO15 activate cdks by phosphorylating the same residue, the structural mechanisms underlying the enzyme-substrate recognition differ greatly. Structural and physiological implications of these findings will be discussed.

Electron Tomography of Metaphase Nucleolar Organizer Regions: Evidence for a Twisted-Loop Organization

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60–80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.

Nuclear Accumulation of S-Adenosylhomocysteine Hydrolase in Transcriptionally Active Cells during Development of Xenopus laevis

The oocyte nuclear antigen of the monoclonal antibody 32-5B6 of Xenopus laevis is subject to regulated nuclear translocation during embryogenesis. It is distributed in the cytoplasm during oocyte maturation, where it remains during cleavage and blastula stages, before it gradually reaccumulates in the nuclei during gastrulation. We have now identified this antigen to be the enzyme S-adenosylhomocysteine hydrolase (SAHH). SAHH is the only enzyme that cleaves S-adenosylhomocysteine, a reaction product and an inhibitor of all S-adenosylmethionine-dependent methylation reactions. We have compared the spatial and temporal patterns of nuclear localization of SAHH and of nuclear methyltransferase activities during embryogenesis and in tissue culture cells. Nuclear localization of Xenopus SAHH did not temporally correlate with DNA methylation. However, we found that SAHH nuclear localization coincides with high rates of mRNA synthesis, a subpopulation colocalizes with RNA polymerase II, and inhibitors of SAHH reduce both methylation and synthesis of poly(A)+ RNA. We therefore propose that accumulation of SAHH in the nucleus may be required for efficient cap methylation in transcriptionally active cells. Mutation analysis revealed that the C terminus and the N terminus are both required for efficient nuclear translocation in tissue culture cells, indicating that more than one interacting domain contributes to nuclear accumulation of Xenopus SAHH.

Assembly of the Nuclear Transcription and Processing Machinery: Cajal Bodies (Coiled Bodies) and Transcriptosomes

We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem–loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.

Computation, prediction, and experimental tests of fitness for bacteriophage T7 mutants with permuted genomes

We created a simulation based on experimental data from bacteriophage T7 that computes the developmental cycle of the wild-type phage and also of mutants that have an altered genome order. We used the simulation to compute the fitness of more than 105 mutants. We tested these computations by constructing and experimentally characterizing T7 mutants in which we repositioned gene 1, coding for T7 RNA polymerase. Computed protein synthesis rates for ectopic gene 1 strains were in moderate agreement with observed rates. Computed phage-doubling rates were close to observations for two of four strains, but significantly overestimated those of the other two. Computations indicate that the genome organization of wild-type T7 is nearly optimal for growth: only 2.8% of random genome permutations were computed to grow faster, the highest 31% faster, than wild type. Specific discrepancies between computations and observations suggest that a better understanding of the translation efficiency of individual mRNAs and the functions of qualitatively “nonessential” genes will be needed to improve the T7 simulation. In silico representations of biological systems can serve to assess and advance our understanding of the underlying biology. Iteration between computation, prediction, and observation should increase the rate at which biological hypotheses are formulated and tested.

Yeast homologues of higher eukaryotic TFIID subunits

In eukaryotic cells the TATA-binding protein (TBP) associates with other proteins known as TBP-associated factors (TAFs) to form multisubunit transcription factors important for gene expression by all three nuclear RNA polymerases. Computer searching of the complete Saccharomyces cerevisiae genome revealed five previously unidentified yeast genes with significant sequence similarity to known human and Drosophila RNA polymerase II TAFs. Each of these genes is essential for viability. A sixth essential gene (FUN81) has previously been noted to be similar to human TAFII18. Coimmunoprecipitation experiments show that all six proteins are associated with TBP, demonstrating that they are true TAFs. Furthermore, these proteins are present in complexes containing the TAFII130 subunit, indicating that they are components of TFIID. Based on their predicted molecular weights, these genes have been designated TAF67, TAF61(68), TAF40, TAF23(25), TAF19(FUN81), and TAF17. Yeast TAF61 is significantly larger than its higher eukaryotic homologues, and deletion analysis demonstrates that the evolutionarily conserved, histone-like domain is sufficient and necessary to support viability.

Regulation of RpoS proteolysis in Escherichia coli: The response regulator RssB is a recognition factor that interacts with the turnover element in RpoS

The degradation of the RpoS (σS) subunit of RNA polymerase in Escherichia coli is a prime example of regulated proteolysis in prokaryotes. RpoS turnover depends on ClpXP protease, the response regulator RssB, and a hitherto uncharacterized “turnover element” within RpoS itself. Here we localize the turnover element to a small element (around the crucial amino acid lysine-173) directly downstream of the promoter-recognizing region 2.4 in RpoS. Its sequence as well as its location identify the turnover element as a unique proteolysis-promoting motif. This element is shown to be a site of interaction with RssB. Thus, RssB is functionally unique among response regulators as a direct recognition factor in ClpXP-dependent RpoS proteolysis. Binding of RssB to RpoS is stimulated by phosphorylation of the RssB receiver domain, suggesting that environmental stress affects RpoS proteolysis by modulating RssB affinity for RpoS. Initial evidence indicates that lysine-173 in RpoS, besides being essential of RpoS proteolysis, may play a role in promoter recognition. Thus the same region in RpoS is crucial for proteolysis as well as for activity as a transcription factor.

Mechanism for a transcriptional activator that works at the isomerization step

Transcriptional activators in prokaryotes have been shown to stimulate different steps in the initiation process including the initial binding of RNA polymerase (RNAP) to the promoter and a postbinding step known as the isomerization step. Evidence suggests that activators that affect initial binding can work by a cooperative binding mechanism by making energetically favorable contacts with RNAP, but the mechanism by which activators affect the isomerization step is unclear. A well-studied example of an activator that normally exerts its effect exclusively on the isomerization step is the bacteriophage λ cI protein (λcI), which has been shown genetically to interact with the C-terminal region of the σ70 subunit of RNAP. We show here that the interaction between λcI and σ can stimulate transcription even when the relevant portion of σ is transplanted to another subunit of RNAP. This activation depends on the ability of λcI to stabilize the binding of the transplanted σ moiety to an ectopic −35 element. Based on these and previous findings, we discuss a simple model that explains how an activator's ability to stabilize the binding of an RNAP subdomain to the DNA can account for its effect on either the initial binding of RNAP to a promoter or the isomerization step.

A cellular defense pathway regulating transcription through poly(ADP-ribosyl)ation in response to DNA damage

DNA damage is known to trigger key cellular defense pathways such as those involved in DNA repair. Here we provide evidence for a previously unrecognized pathway regulating transcription in response to DNA damage and show that this regulation is mediated by the abundant nuclear enzyme poly(ADP-ribose) polymerase. We found that poly(ADP-ribose) polymerase reduced the rate of transcription elongation by RNA polymerase II, suggesting that poly(ADP-ribose) polymerase negatively regulates transcription, possibly through the formation of poly(ADP-ribose) polymerase–RNA complexes. In damaged cells, poly(ADP-ribose) polymerase binds to DNA breaks and automodifies itself in the presence of NAD+, resulting in poly(ADP-ribose) polymerase inactivation. We found that automodification of poly(ADP-ribose) polymerase in response to DNA damage resulted in the up-regulation of transcription, presumably because automodified poly(ADP-ribose) polymerase molecules were released from transcripts, thereby relieving the block on transcription. Because agents that damage DNA damage RNA as well, up-regulation of RNA synthesis in response to DNA damage may provide cells with a mechanism to compensate for the loss of damaged transcripts and may be critical for cell survival after exposure to DNA-damaging agents.