Eukaryotic Transcription Systems Research Articles

AID-RNA polymerase II transcription-dependent deamination of IgV DNA.

Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes to produce high-affinity antibodies. SHM requires IgV transcription by RNA polymerase II (Pol II). A eukaryotic transcription system including AID has not been reported previously. Here, we reconstitute AID-catalyzed deamination during Pol II transcription elongation in conjunction with DSIF transcription factor. C→T mutations occur at similar frequencies on non-transcribed strand (NTS) and transcribed strand (TS) DNA. In contrast, bacteriophage T7 Pol generates NTS mutations predominantly. AID-Pol II mutations are strongly favored in WRC and WGCW overlapping hot motifs (W = A or T, R = A or G) on both DNA strands. Single mutations occur on 70% of transcribed DNA clones. Mutations are correlated over a 15 nt distance in multiply mutated clones, suggesting that deaminations are catalyzed processively within a stalled or backtracked transcription bubble. Site-by-site comparisons for biochemical and human memory B-cell mutational spectra in an IGHV3-23*01 target show strongly favored deaminations occurring in the antigen-binding complementarity determining regions (CDR) compared to the framework regions (FW). By exhibiting consistency with B-cell SHM, our in vitro data suggest that biochemically defined reconstituted Pol II transcription systems can be used to investigate how, when and where AID is targeted.

Improved method for soluble expression and rapid purification of yeast TFIIA

In vitro transcription systems have been utilized to elucidate detailed mechanisms of transcription. Purified RNA polymerase II (pol II) and general transcription factors (GTFs) are required for the in vitro reconstitution of eukaryotic transcription systems. Among GTFs, TFIID and TFIIA play critical roles in the early stage of transcription initiation; TFIID first binds to the DNA in transcription initiation and TFIIA regulates TFIID's DNA binding activity. Despite the important roles of TFIIA, the time-consuming steps required to purify it, such as denaturing and refolding, have hampered the preparation of in vitro transcription systems. Here, we report an improved method for soluble expression and rapid purification of yeast TFIIA. The subunits of TFIIA, TOA1 and TOA2, were bacterially expressed as fusion proteins in soluble form, then processed by the PreScission protease and co-purified. TFIIA's heterodimer formation was confirmed by size exclusion chromatography−multiangle light scattering (SEC−MALS). The hydrodynamic radius (Rh) and radius of gyration (Rg) were measured by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), respectively. The Rg/Rh value implied that the intrinsically disordered region of TOA1 might not have an extended structure in solution. Our improved method provides highly purified TFIIA of sufficient quality for biochemical, biophysical, and structural analyses of eukaryotic transcription systems.

Regulation of a Myb Transcription Factor by Cyclin-dependent Kinase 2 in Giardia lamblia

The protozoan Giardia lamblia parasitizes the human small intestine to cause diseases. It undergoes differentiation into infectious cysts by responding to intestinal stimulation. How the activated signal transduction pathways relate to encystation stimulation remain largely unknown. During encystation, genes encoding cyst wall proteins (CWPs) are coordinately up-regulated by a Myb2 transcription factor. Because cell differentiation is linked to cell cycle regulation, we tried to understand the role of cell cycle regulators, cyclin-dependent kinases (Cdks), in encystation. We found that the recombinant Myb2 was phosphorylated by Cdk-associated complexes and the levels of phosphorylation increased significantly during encystation. We have identified a putative cdk gene (cdk2) by searching the Giardia genome database. Cdk2 was found to localize in the cytoplasm with higher expression during encystation. Interestingly, overexpression of Cdk2 resulted in a significant increase of the levels of cwp gene expression and cyst formation. In addition, the Cdk2-associated complexes can phosphorylate Myb2 and the levels of phosphorylation increased significantly during encystation. Mutations of important catalytic residues of Cdk2 resulted in a significant decrease of kinase activity and ability of inducing cyst formation. Addition of a Cdk inhibitor, purvalanol A, significantly decreased the Cdk2 kinase activity and the levels of cwp gene expression and cyst formation. Our results suggest that the Cdk2 pathway may be involved in phosphorylation of Myb2, leading to activation of the Myb2 function and up-regulation of cwp genes during encystation. The results provide insights into the use of Cdk inhibitory drugs in disruption of Giardia differentiation into cysts.

Structural basis of transcription by bacterial and eukaryotic RNA polymerases

DNA-dependent RNA polymerase (RNAP) is responsible for cellular gene transcription. Although crystallographic studies on prokaryotic and eukaryotic RNAPs have elucidated the basic RNAP architectures, the structural details of many essential events during transcription initiation, elongation, and termination are still largely unknown. Recent crystallographic studies on a bacterial RNAP and yeast RNAP II have revealed different RNAP structural states from that of the normal transcribing complex, as well as the basis of transcription factor functions, advancing our understanding of transcription. These studies have highlighted unexpected similarities in many fundamental aspects of transcription mechanisms between the bacterial and eukaryotic transcription machineries. Remarkable differences also exist between the bacterial and eukaryotic transcription systems, suggesting directions for future studies.

Genetic and transcriptomic analysis of transcription factor genes in the model halophilic Archaeon: coordinate action of TbpD and TfbA

BackgroundArchaea are prokaryotic organisms with simplified versions of eukaryotic transcription systems. Genes coding for the general transcription factors TBP and TFB are present in multiple copies in several Archaea, including Halobacterium sp. NRC-1. Multiple TBP and TFBs have been proposed to participate in transcription of genes via recognition and recruitment of RNA polymerase to different classes of promoters.ResultsWe attempted to knock out all six TBP and seven TFB genes in Halobacterium sp. NRC-1 using the ura3-based gene deletion system. Knockouts were obtained for six out of thirteen genes, tbpCDF and tfbACG, indicating that they are not essential for cell viability under standard conditions. Screening of a population of 1,000 candidate mutants showed that genes which did not yield mutants contained less that 0.1% knockouts, strongly suggesting that they are essential. The transcriptomes of two mutants, ΔtbpD and ΔtfbA, were compared to the parental strain and showed coordinate down regulation of many genes. Over 500 out of 2,677 total genes were regulated in the ΔtbpD and ΔtfbA mutants with 363 regulated in both, indicating that over 10% of genes in both strains require the action of both TbpD and TfbA for normal transcription. Culturing studies on the ΔtbpD and ΔtfbA mutant strains showed them to grow more slowly than the wild-type at an elevated temperature, 49°C, and they showed reduced viability at 56°C, suggesting TbpD and TfbA are involved in the heat shock response. Alignment of TBP and TFB protein sequences suggested the expansion of the TBP gene family, especially in Halobacterium sp. NRC-1, and TFB gene family in representatives of five different genera of haloarchaea in which genome sequences are available.ConclusionSix of thirteen TBP and TFB genes of Halobacterium sp. NRC-1 are non-essential under standard growth conditions. TbpD and TfbA coordinate the expression of over 10% of the genes in the NRC-1 genome. The ΔtbpD and ΔtfbA mutant strains are temperature sensitive, possibly as a result of down regulation of heat shock genes. Sequence alignments suggest the existence of several families of TBP and TFB transcription factors in Halobacterium which may function in transcription of different classes of genes.

Molecular Characterization of a New Evolutionarily Conserved Nuclear Protein e(y)2

Molecular structural and function analyses of the Drosophila melanogasterenhancer of yellow 2 (e(y)2) gene showed that its product acts as a transcription factor and is one of the basic elements of the eukaryotic transcription system. The gene is expressed at all stages of D. melanogaster development and consists of a single intron coding for the protein of 101 amino acid residues. The e(y)2 protein does not contain regions homologous to known proteins. The protein binds with chromatin but not with DNA. On evidence of immune staining, e(y)2 occurs in the nuclei of all D. melanogaster cells. Each nucleus contains approximately 1.2 × 104 molecules of the protein. Immune staining revealed approximately 200 sites of e(y)2 location on polytene chromosomes. The protein is evolutionarily conserved: its homologs were found in evolutionary distant organisms, such as plants, mammals, and protozoans. Amino acid sequences of human, rabbit, and mouse e(y)2 are identical to each other.

Transcriptional regulation of the adenovirus E1A gene.

Two distinct classes of the regulatory sequences have been identified in eukaryotic transcription systems. Promoter sequences are located close to the transcription initiation site, and enhancer sequences can be located far upstream or downstream from the initiation site. Most of the enhancer consists of multiple DNA-binding sites for proteins which act either synergistically or antagonistically to modulate transcriptional activity (Johnson and McKnight 1989; Mitchell and Tjian 1989). The 72-base pair (bp) repeats of the simian virus 40 (SV40) early promoter/enhancer region constitute one of the best-characterized enhancer elements (Herr and Clarke 1986; Zenke et al. 1986; Ondek et al. 1988). The region is composed of a number of modular units, and the combined action of these different units determines the degree of enhancer activity in different cell types. Promoter sequences include the TATA box and initiation sites of transcription, which are involved in the positioning of the transcription initiation site. Promoters of house-keeping genes contain GC-rich sequences and an initiator element instead of the TATA box. These cis-acting elements operate by interacting with specific DNA-binding proteins. Therefore, an understanding of the mechanisms operational in transcriptional regulation of eukaryotic genes entails unravelling the mechanisms controlling the activity of these DNA-binding proteins (McKnight and Yamamoto 1992).

RNA polymerase III dependence of the human L1 promoter and possible participation of the RNA polymerase II factor YY1 in the RNA polymerase III transcription system.

From the general views of the eukaryotic transcription systems, L1 (or L1-like) retrotransposons that encode some proteins are unusual. L1, unlike other protein-coding elements, is transcribed through an internal promoter. And the L1 internal promoter, unlike other internal promoters, is thought to be RNA polymerase II (pol II) dependent, because the L1 transcript has a large size (approximately 6 kb), protein coding capacity and a 3' terminal polyadenylation signal followed by a poly(A) tail, and also because transcription from the promoter of Drosophila L1-like element jockey was highly sensitive to alpha-amanitin. However, our in vitro transcription study reveals that transcription from the human L1 promoter is highly sensitive to tagetitoxin, a selective inhibitor of RNA polymerase III (pol III), but insensitive to 1 micrograms/ml of alpha-amanitin, indicating that the human L1 promoter is pol III-dependent. The pol III dependence is further supported by our observation that L1 and pol III-dependent tRNA gene promoters share a common nuclear factor YY1. There is evidence that YY1 is also a pol II transcription factor. We thus propose that YY1 is a possible member of the pol III transcription system.

Inhibition of prokaryotic and eukaryotic transcription by the 2:1 2,9-dimethyl-1,10-phenanthroline-cuprous complex, a ligand specific for open complexes.

The redox-stable, tetrahedral cuprous chelate of neocuproine (2,9-dimethyl-1,10-phenanthroline) binds to the single-stranded DNA formed in open complexes and is an effective inhibitor of eukaryotic and prokaryotic transcription. Despite the many kinetic and structural differences between prokaryotic and eukaryotic transcription systems, they are all similarly inhibited by neocuproine copper, suggesting that all open complexes may share a homologous structure.

Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II

Using a defined RNA polymerase II (pol II) transcription system, we have investigated the roles of basal factors at discrete stages during the transcription cycle (e.g., initiation, promoter clearance, and transcript elongation). Abortive initiation assays revealed that TATA-binding protein, transcription factors TFIIB and TFIIF, and pol II were necessary and sufficient to form functional initiation complexes on both linear and supercoiled templates. By contrast, TFIIE, TFIIH, and ATP hydrolysis were additionally required during promoter clearance from linear templates, while negative supercoiling obviated the need for these auxiliary factors. Furthermore, TFIIE, TFIIH, and supercoiling were not required during elongation. Our results suggest a role for TFIIH-associated helicase activity or supercoiling during promoter clearance rather than open complex formation. These results establish abortive initiation as a useful assay for studying functional initiation complex formation in defined eukaryotic transcription systems and provide a framework for investigating regulation at different stages of the eukaryotic transcription cycle.

APhaseolus vulgaris mitochondrial tRNALeu is identical to its cytoplasmic counterpart: sequencing andin vivo transcription of the gene corresponding to the cytoplasmic tRNALeu

We report here that the sequence ofP. vulgaris mitochondrial and cytoplasmic tRNA(Leu) (NAA) are identical except for a post-transcriptional modification. There is an unidentified modification at the "wobble" position which, from the sequence of the nuclear tRNA(Leu) gene, we identify as a derivative of C. We also show that thisP. vulgaris nuclear gene is functional by demonstrating its transcription in anin vivo eukaryotic transcription system.

Transcriptional control mechanisms

This book consists of papers presented at a symposium on mechanisms of transcriptional control. Topics discussed include: 5'-regulatory elements of the human HPRT gene;nuclear factors interacting with immunoglobulin heavy and K enhancers;mitochondinal transcription factor binds novel control elements of both major promoters of human mt DNA;RNA processing and translation;chromatin structure and assembly;hormonal regulation of transcription and eukaryotic transcription systems.