Fractions containing the 40S subunit were identified by probing for the small ribosomal protein RpS25

Fractions containing the 40S subunit were identified by probing for the small ribosomal protein RpS25. target genes. The initiation of mRNA transcription involves the assembly of a transcription preinitiation complex (PIC), which as a minimum includes RNA polymerase II (RNAP II), Mediator, and six general transcription factors (TFIIA, -B, -D, -E, -F, and -H) at the core promoter DNA region (23,32,38). PIC assembly is initiated by the binding of the TATA box binding protein (TBP) subunit of TFIID to the promoter, which is stabilized in the presence of TFIIA and Mediator. Subsequently, TFIIB binds to and stabilizes the TFIIA-TFIIB-Mediator-DNA complex and functions as an adaptor that recruits the preformed RNAP II-TFIIF complex to the promoter. TFIIE and TFIIH then join to form the complete PIC. Once the PIC has been assembled on the promoter, transcription initiation occurs in several steps, which involve extensive phosphorylation Phentolamine mesilate of the C-terminal domain (CTD) of RNAP II (9). Early on in the transition Phentolamine mesilate from preinitiation to elongation, phosphorylation of Ser5s in the CTD heptapeptide repeats takes place, and this depends on the activity of the TFIIH-associated kinase cyclin-dependent kinase 7 (Cdk7; mammals)/Kin28 (yeast) (20,42). Subsequently, Ser2s are phosphorylated by the elongation phase kinase Cdk9 (mammals)/CTDK-1 (yeast) to generate elongation-proficient RNAP II complexes (22,28). Another Cdk, Cdk8, can negatively regulate RNAP II transcription, partially via its inhibitory effect on Cdk7 activity (3). More recently, it has been suggested that Cdk11p110regulates RNAP II transcription in humans. Thus, Cdk11p110binds to hypo- and hyperphosphorylated RNAP II (47,52), and antibody-mediated repression of Cdk11p110activity results in inhibition of RNAP II transcription (47). In addition to the phosphorylation events that control RNAP II activity, modification of the chromatin structure represents an important mechanism for regulating gene expression (41). When the chromatin is in its repressed state, the DNA is wrapped tightly around the histones, creating a barrier to the assembly of the RNAP II PIC at the promoter region. Activation of gene expression is associated with a number of histone modifications that loosen the chromatin structure, including acetylation, methylation, ubiquitylation, and phosphorylation (reviewed in reference41). Histone H3 and H4 acetylations are particularly frequent toward the 5 ends of actively transcribed genes and presumably facilitate the initial assembly of the PICs at the promoter region. MOF (males absent on first) is a histone H4 lysine 16 (H4K16)-specific histone acetyltransferase (HAT) in both mammals andDrosophila(2,15,30,45,46). MOF is part of several complexes, including theDrosophilamale-specific lethal (MSL) complex, which is required for X chromosome dosage compensation (2,15,30,45), the mammalian counterpart of the MSL complex (46), and the MOF-MSL1v1 complex, which mediates p53 acetylation at K120 (11,25). In addition, MOF copurifies with a number of other proteins, such as the forkhead-associated (FHA) domain-containing protein MCRS2, NSL1-3 (for nonspecific Phentolamine mesilate lethal 1 to 3), and MBD-R2, as part of the NSL complex (8,30,34,35). In the present study, we focus on the function ofDrosophilaMCRS2 (dMCRS2), theDrosophilaortholog of human MCRS2 (also known as MSP58). Whereas human MCRS1 and -2 proteins have been associated with a variety of cellular processes, including RNA polymerase I transcription (43) and cell cycle progression (16), dMCRS2 Rabbit Polyclonal to TEP1 is largely uncharacterized. In addition to the recent observation that human andDrosophilaMCRS2s form complexes with MOF (8,34,30,35), several other reports suggest that MCRS1 and -2 proteins could function in transcription regulation via interactions with the transcriptional repressor Daxx (27) or the basic region leucine zipper factor Nrf1 (50). We show that dMCRS2 can be affinity purified in complex with several Phentolamine mesilate RNAP II components. Moreover, dMCRS2 associates with RNAP IIin vitroand colocalizes with RNAP II complexes on polytene chromosomesin vivo. Finally, dMCRS2 is required for.