The positive transcription elongation factor b (P-TEFb) plays a pivotal role in productive elongation of nascent RNA molecules by RNA polymerase II. Core active P-TEFb is composed of CDK9 and cyclin T. In addition, mammalian cell extracts contain an inactive P-TEFb complex composed of four components, CDK9, cyclin T, the 7SK snRNA and the MAQ1/HEXIM1 protein. We now report an in vitro reconstitution of 7SK-dependent HEXIM1 association to purified P-TEFb and subsequent CDK9 inhibition. Yeast three-hybrid tests and gel-shift assays indicated that HEXIM1 binds 7SK snRNA directly and a 7SK snRNArecognition motif was identified in the central part of HEXIM1 (amino acids (aa) 152-155). Data from yeast two-hybrid and pull-down assay on GST fusion proteins converge to a direct binding of P-TEFb to the HEXIM1 C-terminal domain (aa 181-359). Consistently, point mutations in an evolutionarily conserved motif (aa 202-205) were found to suppress P-TEFb binding and inhibition without affecting 7SK recognition. We propose that the RNA-binding domain of HEXIM1 mediates its association with 7SK and that P-TEFb then enters the complex through association with HEXIM1.

Positive transcription elongation factor b (P-TEFb) comprises a cyclin (T1 or T2) and a kinase, cyclindependent kinase 9 (CDK9), which phosphorylates the carboxyl-terminal domain of RNA polymerase II. P-TEFb is essential for transcriptional elongation in human cells. A highly specific interaction among cyclin T1, the viral protein Tat, and the transactivation response (TAR) element RNA determines the productive transcription of the human immunodeficiency virus genome. In growing HeLa cells, half of P-TEFb is kinase inactive and binds to the 7SK small nuclear RNA. We now report on a novel protein termed MAQ1 (for ménage à quatre) that is also present in this complex. Since 7SK RNA is required for MAQ1 to associate with P-TEFb, a structural role for 7SK RNA is proposed. Inhibition of transcription results in the release of both MAQ1 and 7SK RNA from P-TEFb. Thus, MAQ1 cooperates with 7SK RNA to form a novel type of CDK inhibitor. According to yeast two-hybrid analysis and immunoprecipitations from extracts of transfected cells, MAQ1 binds directly to the N-terminal cyclin homology region of cyclins T1 and T2. Since Tat also binds to this cyclin T1 N-terminal domain and since the association between 7SK RNA/MAQ1 and P-TEFb competes with the binding of Tat to cyclin T1, we speculate that the TAR RNA/Tat lentivirus system has evolved to subvert the cellular 7SK RNA/MAQ1 system. Phosphorylation of the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) is a critical step required for transcription elongation (7) and for recruitment of the machinery involved in pre-mRNA maturation (3,26,46). The CTD is unphosphorylated when RNAP II assembles onto promoters (RNAP IIA). A class of negative transcription factors including the 5,6-dichlorozo-1-␤-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor and the negative elongation factor causes transcriptional arrest shortly after initiation, during which the polymerase may fall off (60). To release this block, the CTD must be phosphorylated (RNAP IIO) by positive transcription elongation factor b (P-TEFb), a protein complex that comprises cyclin-dependent kinase 9 (CDK9) and a cyclin (T1 or T2) (45). P-TEFb kinase activity is required for transcription of most class II genes (6).The human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter uses a unique mechanism: the level of proviral DNA transcription is determined by recruitment of P-TEFb to the TAR (transactivation response) element, an RNA stem-loop structure that forms at the 5Ј end of the viral transcript (4,38,59,66). The viral genome encodes a very potent transactivator of its own transcription, the Tat protein.The formation of a quaternary complex among CDK9, cyclin T1, Tat, and TAR RNA determines the recruitment of human P-TEFb to the transcription elongation complex and the efficient synthesis of long productive viral transcripts (15,18,30,33,44,65).Binding of the 7SK small nuclear RNA (snRNA) to P-TEFb has recently been shown to be associated with the inhibition of CDK9 kinase activity...

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The positive transcription elongation factor P-TEFb controls the elongation of transcription by RNA polymerase II. P-TEFb is inactivated upon binding to HEXIM1 or HEXIM2 proteins associated with a noncoding RNA, 7SK. In response to the inhibition of transcription, 7SK RNA, as well as HEXIM proteins, is released by an unknown mechanism and P-TEFb is activated. New partners of 7SK RNA were searched for as potential players in this feedback process. A subset of heterogeneous ribonuclear proteins, hnRNPs Q and R and hnRNPs A1 and A2, were thus identified as major 7SK RNA-associated proteins. The degree of association of 7SK RNA with these hnRNPs increased when P-TEFb-HEXIM1-7SK was dissociated following the inhibition of transcription or HEXIM1 knockdown. This finding suggested that 7SK RNA shuttles from HEXIM1-P-TEFb complexes to hnRNPs. The transcription-dependent dissociation of P-TEFb-HEXIM1-7SK complexes was attenuated when both hnRNPs A1 and A2 were knocked down by small interfering RNA. As hnRNPs are known to interact transiently with RNA while it is synthesized, hnRNPs released from nascent transcripts may trap 7SK RNA and thereby contribute to the activation of P-TEFb.The positive transcription elongation factor P-TEFb is required to activate the transcription of most class II genes (48). P-TEFb comprises two subunits, CDK9, a cyclin-dependent protein kinase, and its corresponding cyclin T1 or cyclin T2. The activity of P-TEFb is regulated. It increases in response to the inhibition of transcription (45, 63) or cardiac cell hypertrophic stimulation (54). Previous studies have indicated that 7SK RNA associates with an inactive form of P-TEFb. 7SK RNA is an abundant (2 ϫ 10 5 -molecule-per-cell) noncoding nuclear RNA of 331 nucleotides (60, 68). The inhibition of P-TEFb activity relies upon the binding of HEXIM1 or HEXIM2 proteins to cyclin T1 or T2 (5,13,41,64). This process requires the association of 7SK RNA with HEXIM1 or HEXIM2 (40,65). Two hairpins in the 7SK RNA structure are involved in this association (14). 7SK binding to HEXIM proteins promotes a major conformational change allowing the C-terminal domains of the proteins to interact with the Nterminal domains of cyclin T's (41, 55). This transcriptiondependent regulation may constitute a feedback loop finetuning the efficiency of the elongation step in class II gene transcription.The molecular regulatory mechanism remains largely unknown. 7SK RNA is very stable even when it dissociates from HEXIM proteins; its degradation is unlikely to contribute to the dissociation. Posttranslational modifications of protein subunits in P-TEFb-HEXIM-7SK may determine their association. For instance, the phosphorylation of CDK9 on threonine residue T186 is required, but the in vivo regulation of this step has not been established (10,35,47). HEXIM proteins and 7SK RNA may be released when P-TEFb binds to components of the transcriptional machinery, such as transcription factors like NF-B (2), retinoblastoma protein (56), androgen receptor (34), aryl hydrocarbon r...

Binding region and link protein were prepared from pig laryngeal cartilage proteoglycans after chondroitinase ABC and trypsin digestion. Experiments on gel chromatography showed the purified binding region to interact reversibly with hyaluronate (HA), and this binding was also shown to be stabilized by native link protein. The trypsin-prepared link protein showed properties of self-association in solution that were partially inhibited by oligosaccharides (HA10-16) and abolished by modification of free amino groups (lysine residues) with 2-methylmaleic anhydride. The Mr (sedimentation equilibrium) of the modified link protein was 41 700. Analysis of binding region showed it to contain 25% (w/w) carbohydrate, mainly in galactose, glucosamine, mannose and galactosamine. It contained some keratan sulphate, as digestion with endo-beta-D-galactosidase (keratanase) removed 28% galactose and 25% glucosamine and the Mr (sedimentation equilibrium) decreased from 66 500 to 60 800. After keratanase digestion the interaction with polyclonal antibodies specific for binding region was unaffected, but the response in a radioimmunoassay with a monoclonal antibody to keratan sulphate was decreased by 47%. Preparation of a complex between binding region, link protein and HA approximately 34 showed a single component (5.5S) of Mr (sedimentation equilibrium) 133 500. In this complex the antigenic determinants of link protein appeared masked, as previously found with proteoglycan aggregates. The isolated binding region and link protein were thus shown to retain properties comparable with those involved in the structure and organization of proteoglycan aggregates.

The molecular characterization of a human testicular proteoglycan, the progenitor of a seminal plasma glycosaminoglycan-bearing peptide, was achieved by cDNA cloning, Its protein core encompasses several domains encountered in various proteins associated with adhesion, migration and cell proliferation. An osteonectin-like domain, a Kazal-like sequence and a 46-amino-acid motif around a Cys-Trp-Cys-Val peptide encountered in cell-surface antigens, cell-adhesion molecules and growth-factor-binding proteins are distributed within the testican protein core. Testican is the progenitor of the unique heparadchondroitin-sulfate-bearing peptide present in human seminal plasma, a feature which might confer additional potentialities to this hybrid proteoglycan.Proteoglycans belong to a versatile protein family (Ruoslahti, 1988) whose potential functions proceed from either the glycosaminoglycan chains they bear or from specific regions of their protein cores (Wight et al., 1991 ; Kjelltn and Lindahl, 1991); they contribute to maintain an essential microenvironment for cell adhesion, migration and proliferation (Wight et al., 1992) through their ability to function as linkers between cells and the extracellular matrix (ECM) and as growth-factor binders (Ruoslahti, 1989;Ruoslahti and Yamaguchi, 1991). ECM components of the testis regulate, in vitro, Sertoli-cell differentiation, testicular-cord formation and germ-cell development, mimicking the original testicular structure (Hadley et al., 1985). Among the components of testicular ECM, only proteoglycans synthesized by rat peritubular cells and Sertoli cells in culture have so far been partially characterized ; while peritubular cells only produce proteochondroitin, Sertoli cells produce proteochondroitin, proteoheparan and a hybrid proteoglycan (Skinner and Fritz, 1985) bearing both chondroitin (CS) and heparan-sulfate (HS) chains. On the basis of the recent isolation of a single and unique glycosaminoglycan-bearing peptide (Bonnet et al., 1992) from human seminal plasma, (S.GP), substituted with both HS and CS, the characterization of its progenitor, speculated to be synthesized in the testis, was achieved by cDNA cloning. Abbreviations. S.GP, glycosaminoglycan-bearing peptide from human seminal plasma; RA-S.GP, reduced and alkylated glycosaminoglycan-bearing peptide from human seminal plasma; ECM, extracellular matrix; CS, chondroitin-sulfate chain; HS heparan-sulfate chain. EXPERIMENTAL PROCEDURES6 X lo5 clones of a human testicular DNA library in Agtll (Clontech) were screened with a 38-nucleotide ~'~P-labelled probe (5'-GTCTCCTGCTCCTCGCAGGACACGGCGCCC-TGCTT-3'), corresponding to the first 13 amino acids identified in the purified reduced and alkylated form of S.GP (RA-S.GP; Bonnet et al., 1992). Hybond N+ (Amersham) filter hybridization was carried out in 6 X NaCVCit. (0.15 M NaCI, 0.015 M sodium citrate), 5 X Denhardt, 0.5% SDS and 250 pg/ml salmon-sperm DNA for 18 h at 42°C. The filters were washed five times at 48°C for 10 min in 4 X NaCKit. and subjected to autorad...

Campylobacter pyloridis" has been recently described as a gastritis-associated bacterium. We studied 20 strains. The bacteria had most of the characteristics of Campylobacter spp. strains. They were hippurate negative and tolerant to triphenyl tetrazolium chloride (at 0.4 and 1 mg/ml). They grew on all the media commonly used in laboratories, although chocolate agar was the most effective for isolation. They grew in a microaerophilic atmosphere as well as in an atmosphere enriched in C02 and when incubated at 37°C but not at 30 or 42°C. A total of 31 enzymes were present among the 78 studied. y-Glutamyl-transpeptidase activity was, in addition to urea hydrolysis, an interesting feature for the identification of these bacteria. The protein profiles of the 20 strains were similar.

The positive transcription elongation factor (P-TEFb) comprises a kinase, CDK9, and a Cyclin T1 or T2. Its activity is inhibited by association with the HEXIM1 or HEXIM2 protein bound to 7SK small nuclear RNA. HEXIM1 and HEXIM2 were found to form stable homoand hetero-oligomers. Using yeast two-hybrid and transfection assays, we have now shown that the C-terminal domains of HEXIM proteins directly interact with each other. Hydrodynamic parameters measured by glycerol gradient ultracentrifugation and gel-permeation chromatography demonstrate that both purified recombinant and cellular HEXIM1 proteins form highly anisotropic particles. Chemical cross-links suggest that HEXIM1 proteins form dimers. The multimeric nature of HEXIM1 is maintained in P-TEFb⅐HEXIM1⅐7SK RNA complexes. Multiple P-TEFb modules are found in the inactive P-TEFb⅐HEXIM1⅐7SK complexes. It is proposed that 7SK RNA binding to a HEXIM1 multimer promotes the simultaneous recruitment and hence inactivation of multiple P-TEFb units.The positive transcription elongation factor (P-TEFb) 1 comprises a protein kinase, CDK9, and a Cyclin T1, T2, or K (1, 2). It is required for transcription elongation of most class II genes. P-TEFb phosphorylates numerous substrates, including the C-terminal domain (CTD) of RNA polymerase II and the Spt5 subunit of the DRB sensitivity-inducing factor (DSIF). The DSIF prevents transcription from proceeding efficiently after initiation. The kinase activity of P-TEFb antagonizes this inhibitory effect. Several class II genes such as heat-shock or U2 small nuclear RNA genes do not have a strong requirement for P-TEFb activity to elongate, but rather to terminate, transcription properly (3, 4).Recent studies have indicated that two major forms of PTEFb are present in equivalent amounts in HeLa cell lysates (5). The active form consists of "core" P-TEFb, CDK9 and Cyclin T1 or T2. The inactive form consists of CDK9, Cyclin T1 or T2, MAQ1/HEXIM1, and 7SK RNA (5-8). The 7SK RNA is an abundant class III noncoding RNA (9) detected in vertebrates, cephalochordates, and mollusks.2 Binding of 7SK RNA to HEXIM1 turns this protein into a P-TEFb inhibitor (10). In response to treatments that arrest transcription (5-8) or following cardiac hypertrophic stimuli (11, 12), HEXIM1 and 7SK RNA dissociate from P-TEFb. As a consequence, the P-TEFb activity is up-regulated.HEXIM1 expression is up-regulated in smooth muscle cells treated with hexamethylene-bisacetamide (13) or down-regulated by estrogen in breast cancer cells and therefore named EDG-1 (14). HEXIM1 has also been reported as a protein accumulating in heart tissues during early embryogenesis and therefore named CLP-1 (cardiac lineage protein) (15). Disruption of the HEXIM1/CLP1 gene results in heart defects leading to death at birth in homozygote CLP-1(Ϫ/Ϫ) mice (16). Because P-TEFb plays a general role in class II gene expression, such a late developmental defect suggests the existence of a compensatory mechanism at the cellular level. Indeed, a BLAST search within genome and Express...