Mycobacterium tuberculosis infects one-third of the world's human population. This widespread infection depends on the organism's ability to escape host defenses by gaining entry and surviving inside the macrophage. DNA sequences of M. tuberculosis have been cloned; these confer on a nonpathogenic Escherichia coli strain an ability to invade HeLa cells, augment macrophage phagocytosis, and survive for at least 24 hours inside the human macrophage. This capacity to gain entry into mammalian cells and survive inside the macrophage was localized to two distinct loci on the cloned M. tuberculosis DNA fragment.

Cyclin-dependent kinase (CDK)7-cyclin H, the CDK-activating kinase (CAK) and TFIIH-associated kinase in metazoans can be activated in vitro through T-loop phosphorylation or binding to the RING finger protein MAT1. Although the two mechanisms can operate independently, we show that in a physiological setting, MAT1 binding and T-loop phosphorylation cooperate to stabilize the CAK complex of Drosophila. CDK7 forms a stable complex with cyclin H and MAT1 in vivo only when phosphorylated on either one of two residues (Ser164 or Thr170) in its T-loop. Mutation of both phosphorylation sites causes temperature-dependent dissociation of CDK7 complexes and lethality. Furthermore, phosphorylation of Thr170 greatly stimulates the activity of the CDK7- cyclin H-MAT1 complex towards the C-terminal domain of RNA polymerase II without significantly affecting activity towards CDK2. Remarkably, the substrate-specific increase in activity caused by T-loop phosphorylation is due entirely to accelerated enzyme turnover. Thus phosphorylation on Thr170 could provide a mechanism to augment CTD phosphorylation by TFIIH-associated CDK7, and thereby regulate transcription.

Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the metazoan CDK-activating kinase (CAK), which activates CDKs, such as CDC2 and CDK2, through phosphorylation of a conserved threonine residue in the T loop. Full activation of CDK7 requires association with a positive regulatory subunit, cyclin H, and phosphorylation of a conserved threonine residue at position 170 in its own T loop. We show that threonine-170 of CDK7 is phosphorylated in vitro by its targets, CDC2 and CDK2, which also phosphorylate serine-164 in the CDK7 T loop, a site that perfectly matches their consensus phosphorylation site. In contrast, neither CDK4 nor CDK7 itself can phosphorylate the CDK7 T loop in vitro. The ability of CDC2 or CDK2 and CDK7 to phosphorylate each other but not themselves implies that each kinase can discriminate among closely related sequences and can recognize a substrate site that diverges from its usual preferred site. To understand the basis for this paradoxical substrate specificity, we constructed a chimeric CDK with the T loop of CDK7 grafted onto the body of CDK2. Surprisingly, the hybrid enzyme, CDK2-7, was efficiently activated in cyclin A-dependent fashion by CDK7 but not at all by CDK2. CDK2-7, moreover, phosphorylated wild-type CDK7 but not CDK2. Our results suggest that the primary amino acid sequence of the T loop plays only a minor role, if any, in determining the specificity of cyclin-dependent CAKs for their CDK substrates and that protein-protein interactions involving sequences outside the T loop can influence substrate specificity both positively and negatively.Full activation of cyclin-dependent kinases (CDKs) requires the binding of a positive regulatory subunit or cyclin and the phosphorylation of a threonine residue on a conserved loopthe activation segment or T loop-of the catalytic subunit by a CDK-activating kinase (CAK) (reviewed in references 20 and 32). The major CAK in metazoan cells is itself a CDK containing CDK7 as its catalytic subunit (10,29,34,45). That CDK7 activates CDKs in vivo was confirmed in Drosophila melanogaster; flies with a temperature-sensitive CDK7 had a defect in CDC2 phosphorylation, resulting in a block to mitosis at the nonpermissive temperature (25).The T-loop region of CDK7 has a threonine residue, threonine-170 (T170), in the same location as and within a sequence context similar to that for the activating threonines of other CDKs. T170 is required for activation of dimeric complexes of CDK7 with its physiologic partner cyclin H in vitro and in vivo (13, 27) and for the basal kinase activity associated with monomeric CDK7 (28), probably reflecting the phosphorylation of T170 by a putative CAK-activating kinase (CAKAK) (12). What regulatory function, if any, T-loop phosphorylation of CDK7 serves in vivo is unknown. The major form of CDK7 in the cell is a ternary complex of CDK7, cyclin H, and the RING finger protein MAT1 (7,12,47). MAT1 stabilizes the cyclin H-CDK7 complex and can bypass the need for T170 phosphorylation in vitro; trimeric CDK7-cyclin ...

Cyclin-dependent kinase 7 (CDK7) can be isolated as a subunit of a trimeric kinase complex functional in activation of the mitotic promoting factor. In this study, we demonstrate that the trimeric cdk-activating kinase (CAK) acts as a transcriptional repressor of class II promoters and show that repression results from CAK impeding the entry of RNA polymerase II and basal transcription factor IIF into a competent preinitiation complex. This repression is independent of CDK7 kinase activity. We find that the p36/MAT1 subunit of CAK is required for transcriptional repression and the repression is independent of the promoter used. Our results demonstrate a central role for CAK in regulation of messenger RNA synthesis by either inhibition of RNA polymerase II-catalyzed transcription or stimulation of transcription through association with basal transcription repair factor IIH.Cyclin-dependent kinase 7 (CDK7) 1 was originally isolated as the catalytic subunit of the trimeric cdk-activating kinase (CAK) complex. This complex, consisting of CDK7, cyclin H, and MAT1, is responsible for activation of the mitotic promoting factor in vitro (1-3). The discovery that CDK7 was also a component of the basal transcription repair factor IIH (TFIIH) implicated a dual role for CDK7 in transcription as part of TFIIH and in the control of the cell cycle as the trimeric CAK complex (4 -7). TFIIH is a multisubunit protein complex identified as a factor required for RNA polymerase II (RNAPII)-catalyzed transcription (8 -11), and subsequently this complex was found to play a key role in nucleotide excision repair (12)(13)(14). At least nine polypeptides with molecular masses of 89, 80, 62, 52, 44, 40, 37, 36, and 34 kDa co-purify with mammalian TFIIH. The cDNAs encoding all of these subunits have now been cloned. p89 and p80 are the gene products of ERCC3 (XPB) and ERCC2 (XPD), respectively (13-15). p62 and p44 are the mammalian counterparts of the yeast TFB1 and SSL1 gene products that are required for DNA nucleotide excision repair (16). p34 exhibits partial sequence homology to p44 and also contains zinc-finger motifs (17). The p40, p37, and p36 subunits of TFIIH are identical to the vertebrate CAK complexes, CDK7, cyclin H, and p36/MAT1, respectively (5-7). Two subcomplexes containing some TFIIH polypeptides can also be isolated from extracts of HeLa cells (18,19): (i) a five-subunit core TFIIH complex that includes ERCC3 (XPB), p62, p52, p44, and p34 but is devoid of detectable levels of ERCC2 (XPD) or CAK; (ii) an XPD⅐CAK complex that includes XPD and all three CAK components (CDK7, cyclin H, and p36/MAT1). The addition of XPD⅐CAK to the core TFIIH potently stimulates the TFIIH transcriptional activity (18, 19). These observations suggest that core TFIIH and XPD⅐CAK interact to form a complex that constitutes the TFIIH holoenzyme (holo-TFIIH). Biochemical analysis has therefore revealed that CDK7 is a component of at least three complexes, the trimeric CAK complex (20 -22), the quaternary complex with the XPD, and the nine-subunit...