A novel TATA-binding protein-binding protein, ABT1, activates basal transcription and has a yeast homolog that is essential for growth.

Identification of a novel mouse nuclear protein termed activator of basal transcription 1 (mABT1) that associates with the TATA-binding protein (TBP) and enhances basal transcription activity of class II promoters is described. We also identify mABT1 homologous counterparts in Caenorhabditis elegans and Saccharomyces cerevisiae and show the homologous yeast gene to be essential for growth. The mABT1 associated with TBP in HeLa nuclear extracts and with purified mouse TBP in vitro. In addition, ectopically expressed mABT1 was coimmunoprecipitated with endogenous TBP in transfected cells. More importantly, mABT1 significantly enhanced transcription from an adenovirus major late promoter in a reconstituted cell-free system. We furthermore demonstrate that mABT1 consistently enhanced transcription from a reporter gene with a minimal core promoter as well as from reporter genes with various enhancer elements in a cotransfection assay. Taken together, these results suggest that mABT1 is a novel TBP-binding protein which can function as a basal transcription activator.

TATA-Binding protein-interacting protein 120, TIP120, stimulates three classes of eukaryotic transcription via a unique mechanism.

We previously identified a novel TATA-binding protein (TBP)-interacting protein (TIP120) from the rat liver. Here, in an RNA polymerase II (RNAP II)-reconstituted transcription system, we demonstrate that recombinant TIP120 activates the basal level of transcription from various kinds of promoters regardless of the template DNA topology and the presence of TFIIE/TFIIH and TBP-associated factors. Deletion analysis demonstrated that a 412-residue N-terminal domain, which includes an acidic region and the TBP-binding domain, is required for TIP120 function. Kinetic studies suggest that TIP120 functions during preinitiation complex (PIC) formation at the step of RNAP II/TFIIF recruitment to the promoter but not after the completion of PIC formation. Electrophoretic mobility shift assays showed that TIP120 enhanced PIC formation, and TIP120 also stimulated the nonspecific transcription and DNA-binding activity of RNAP II. These lines of evidence suggest that TIP120 is able to activate basal transcription by overcoming a kinetic impediment to RNAP II/TFIIF integration into the TBP (TFIID)-TFIIB-DNA-complex. Interestingly, TIP120 also stimulates RNAP I- and III-driven transcription and binds to RPB5, one of the common subunits of the eukaryotic RNA polymerases, in vitro. Furthermore, in mouse cells, ectopically expressed TIP120 enhances transcription from all three classes (I, II, and III) of promoters. We propose that TIP120 globally regulates transcription through interaction with basal transcription mechanisms common to all three transcription systems.

A notable example of an evolutionary conserved gene: studies on a putative DNA helicase TIP49.

TIP49a (just called as simply TIP49 in previous reports [Kanemaki et al., 1997; Makino et al., 1998]) was found in a rat nuclear protein complex that included the TATA-binding protein. TIP49a possesses multiple sequence motifs for ATPase and DNA helicase. Since TIP49a structurally resembles prokaryotic DNA helicase RuvB, TIP49a is resumed to be a putative DNA helicase. We demonstrated TIP49a-related gene(s) in variety organisms from human to archaea. Amino acid identities expressed as aligned scores of human, yeast, and A. fulgidus TIP49a gene counterparts to the rat sequence were 99, 67, and 46, respectively. Strikingly, two homologous regions of mammalian TIP49a and bacterial RuvB exhibited an aligned score of 17-38. We demonstrated that the eukaryotic TIP49a counterparts were immunologically conserved. These lines of evidence show that the TIP49a gene is a notable example of a highly conserved gene among organisms. An extensive homology search revealed another class of TIP49-related gene in the eukaryotes, designated as TIP49b. Moreover, a phylogenetical study suggested that archaeal TIP49 genes belong to the TIP49b ancestor but not to the TIP49a one and that TIP49a evolved from TIP49b in accordance with divergence of archaea and eukarya. The TIP49 gene family is thought to play a fundamental role in a biological activity.

Provirus of M7 baboon endogenous virus: nucleotide sequence of the gag-pol region.

A 3,023-base nucleotide sequence of the M7 baboon endogenous virus genome, spanning the 5' noncoding region as well as the entire gag gene and part of the pol gene, is reported. Within the 562-base 5' noncoding region, a 21-base sequence complementary to the OH terminus of tRNApro is located immediately downstream from the long terminal repeat. Amino acid sequences were deduced from the 1,596 nucleotides comprising the gag gene, and the four structural gag polypeptides, p12, p15, p30, and p10, appeared to be coded contiguously. Only one termination codon interrupted the M7 gag and pol genes. The data suggest that 55 additional amino acids may be attached to the NH2 terminus of the gag precursor protein. However, such a sequence was not detected in virions or in virus-infected cells. With the exception of the p15 region, nucleotide and amino acid sequences of the gag and pol regions of M7 virus exhibited strong homologies to those of Moloney leukemia virus.

The characteristics of the RNA-dependent DNA polymerase of baboon endogenous virus.

The characteristics of the virion-associated RNA-dependent DNA polymerase (RDPase) of a baboon endogenous virus, M7, were studied extensively; the optimal conditions for the exogenous RDPase reaction were obtained with 0.4 mM-Mn2+, 110 mM-NaCl, 24 microgram/ml poly(rA). oligo(dT)12-18, at pH 7.6 in the presence of 0.01 % Brij-58. Under these conditions, the incorporation of 3H-TMP proceeded up to 90 min at a speed of 0.1 pmol TMP/microgram virus protein/min. Poly(rC). oligo (dG)12-18 and poly(rCm). oligo (dG)12-18 served as the template-primers in the exogenous reaction with 3 mM-Mg2+ and 0.4 mM-Mn2+, respectively. Polyuridylic acid, bleomycin, rifampicin, spermidine and inorganic phosphate significantly inhibited the RDPase activity of BaEV. The RDPase of BaEV requires a higher concentration of NaCl, a lower pH and milder conditions of detergent treatment than those of other mammalian retroviruses.

Identification of a mouse TBP-like protein (TLP) distantly related to the drosophila TBP-related factor.

TATA-binding protein (TBP) is an essential factor for eukaryotic transcription. In this study, we demonstrated a mouse cDNA encoding a 21 kDa TBP-like protein (TLP). The TLP ORF, carrying 186 amino acids, covered the entire 180 amino acids of the C-terminal conserved domain of mouse TBP with 39% identity and 76% similarity. Northern blot analysis demonstrated that TLP mRNAs were expressed in various mammalian tissues ubiquitously and that their distribution pattern was analogous to that of TBP. By using anti-TLP antibody, we demonstrated the existence of TLP proteins in various mammalian cells and tissues. The Drosophila TBP-related factor (TRF) is a neurogenesis-related transcription factor that binds to the TATA-box and activates transcription. TLP did not bind to the TATA-box nor direct transcription initiation. Multiple amino acids critical for TBP function were deleted or substituted in TLP, while amino acids in Drosophila TRF much resembled those in TBP. Similarity between Drosophila TRF and mouse TLP was considerably lower (alignment score 35) than that between Drosophila TBP and mouse TBP (alignment score 88). Identity of nucleotide sequences between mouse and putative human TLPs (94%) was higher than that between TBPs (91%) in these two animals. Expression of TLP was nearly constant throughout the P19 differentiation process. Accordingly, we suggest that, even if higher eukaryotes generally contain multiple tbp -related genes, TLP is not a bona fide mammalian counterpart of Drosophila TRF.

Tissue and cell distribution of a mammalian proteasomal ATPase, MSS1, and its complex formation with the basal transcription factors.

The proteasome is an eukaryotic multi-subunit protease complex composed of one 20S core component and two 19S regulatory complexes. The regulatory complex contains 6 putative ATPases. We investigated tissue and cell distribution of one of these ATPases, MSS1 (mammalian suppressor of sgv1). MSS1 was ubiquitously present in rat tissues as was the 20S core component of proteasome. However, the ratio of MSS1 to 20S varied greatly among tissues and MSS1 was concentrated in the thymus. Glycerol gradient sedimentation analysis revealed that MSS1 is included in protein complexes whose density is lighter than that of the proteasome. MSS1 was distributed in mammalian cells ubiquitously, while proteasome was rather concentrated in the nuclei. Hence, a novel molecular status of MSS1 distinct from proteasome is implicated. Interestingly, multiple basal transcription factors for RNA polymerase II, including TBP, TFIIB, TFIIH, and TFIIF, were found to be associated with MSS1. These results suggest that MSS1, in addition to proteolysis, plays a role in DNA metabolism including transcriptional regulation.

TBP-like protein (TLP/TLF/TRF2) artificially recruited to a promoter stimulates basal transcription in vivo.

Metazoan genomes generally contain one TBP-related gene designated as TBP-like protein (TLP/TLF/TRF2). Although TLP is thought to work for transcriptional regulation, its natural function has not been clearly demonstrated. Here we describe the stimulation of transcription from TATA-containing and TATA-less class II promoters by artificially recruited mammalian TLP. TLP fused with Gal4 DNA-binding domain stimulated transcription when it was recruited at a proximal promoter. Compared to TBP, stimulation by TLP was less TATA-dependent. Slight truncation from each terminus of TLP destroyed this function drastically. Amino acid substitutions of TLP whose corresponding residues in TBP are crucial for its function resulted in the loss of function. Consequently, Gal4-fused TLP was demonstrated to exhibit ability of transcription activation irrespective of the type of promoter, the mechanism of which was thought to be similar to that of artificially recruited TBP. TLP is presumably able to behave as a transcriptional activator in cells.

Regulation of brain-specific transcription of the mouse myelin basic protein gene: function of the NFI-binding site in the distal promoter.

We investigated brain-specific transcription elements in the distal region (-253 to -54) of the mouse myelin basic protein (MBP) promoter by in vitro transcription using mouse brain nuclear extracts. Using deletion and base substitution mutants, we identified one tissue-specific transcription element at the downstream core of the NFI-site between -115 and -111. Foot-printing assay demonstrated that three transcription factors bind around this element; NFI-related (-130 to -111), M1 (-110 to -97) and Sp1 (-92 to -84). NFI-related factor(s) in brain extracts consisted of a characteristic population which was different from that in other tissues. Methylation interference experiments revealed that this factor(s) interacted with the NFI-downstream core in a brain-specific manner. We suggest the existence of a brain-specific NFI-related factor(s).

A rat RuvB-like protein, TIP49a, is a germ cell-enriched novel DNA helicase.

We have isolated a novel nuclear protein with a molecular mass of 49 kDa (TIP49a) from rat liver. The rat TIP49a showed structural resemblance to several bacterial RuvBs and also displayed Walker A and B motifs. We overproduced the recombinant TIP49a in Escherichia coli and purified it to near homogeneity. Biochemical investigations demonstrated that TIP49a possessed ATPase activity that was stimulated by single-stranded DNA but neither by double-stranded DNA nor by any forms of RNA polymers tested. Moreover, a UV cross-linking assay indicated TIP49a specifically interacted with ATP. Interestingly, we found that DNA duplex was unwound by the recombinant TIP49a in the presence of ATP or dATP. Optimal concentrations of ATP and Mg2+ for the helicase activity were 1-2 mM and 0.25-1 mM, respectively. Displacement of the DNA strand occurred in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex. Western blot analysis revealed that TIP49a was abundantly expressed in testes and moderately in spleen, thymus, and lung. In mouse seminiferous tubules, the protein was restrictively observed in germ lineages from late pachytene spermatocytes to round spermatids. From these observations, we propose that TIP49a is a novel DNA helicase and may play a role in nuclear processes such as recombination and transcription.