Brd4 is a mammalian bromodomain protein that binds to acetylated chromatin. Proteomic analysis revealed that Brd4 interacts with cyclinT1 and Cdk9 that constitutes core positive transcription elongation factor b (P-TEFb). Brd4 interacted with P-TEFb in the living nucleus through its bromodomain. About half of P-TEFb was bound to the inhibitory subunit and functionally inactive. Brd4 interacted with P-TEFb that was free of the inhibitory subunit. An increase in Brd4 expression led to increased P-TEFb-dependent phosphorylation of RNA polymerase II (RNAPII) CTD and stimulation of transcription from promoters in vivo. Conversely, a reduction in Brd4 expression by siRNA reduced CTD phosphorylation and transcription, revealing that Brd4 is a positive regulatory component of P-TEFb. In chromatin immunoprecipitation (ChIP) assays, the recruitment of P-TEFb to a promoter was dependent on Brd4 and was enhanced by an increase in chromatin acetylation. Together, P-TEFb alternately interacts with Brd4 and the inhibitory subunit to maintain functional equilibrium in the cell.
The composition of RNase H2 has been a long-standing problem. Whereas bacterial and archaeal RNases H2 are active as single polypeptides, the Saccharomyces cerevisiae homolog, Rnh2Ap, when expressed in Escherichia coli, fails to produce an active RNase H2. By affinity chromatography purification and identification of polypeptides associated with a tagged S.cerevisiae Rnh2Ap, we obtained a complex of three proteins [Rnh2Ap (Rnh201p), Ydr279p (Rnh202p) and Ylr154p (Rnh203p)] that together are necessary and sufficient for RNase H2 activity [correction]. Deletion of the gene encoding any one of the proteins or mutations in the catalytic site in Rnh2A led to loss of RNase H2 activity. Even when S.cerevisiae RNase H2 is catalytically compromised, it still exhibits a preference for cleavage of the phosphodiester bond on the 5' side of a ribonucleotide-deoxyribonucleotide sequence in substrates mimicking RNA-primed Okazaki fragments or a single ribonucleotide embedded in a duplex DNA. Interestingly, Ydr279p and Ylr154p have homologous proteins only in closely related species. The multisubunit nature of S.cerevisiae RNase H2 may be important both for structural purposes and to provide a means of interacting with other proteins involved in DNA replication/repair and transcription.
The rnf genes of Rhodobacter capsulatus, essential for nitrogen fixation, are thought to encode a system for electron transport to nitrogenase. In the present study, we have attempted to overexpress the rnf genes in Escherichia coli to investigate the molecular properties of the corresponding proteins. Corrections were made to the published DNA sequence of the rnf operon, resulting in the identification of two genes, rnfG and rnfH. The rnfABCDGEH operon thus comprises seven genes and shows similarities in gene arrangement and deduced protein sequences to homologous regions in the genomes of Haemophilus influenzae and E. coli. Four of the rnf gene products were found to be similar in sequence to components of an Na ϩ -dependent NADH:ubiquinone oxidoreductase from Vibrio alginolyticus. Three of the rnf genes were successfully overexpressed in E. coli as His-tagged polypeptides, whereas the products of rnfA, rnfD and rnfE, predicted to be transmembrane proteins, could not be stably maintained in E. coli. The rnfB and rnfC gene products were isolated as two brown proteins with apparent molecular-mass values of 25 kDa and 55 kDa, respectively. RnfB was shown to contain one [2Fe-2S] cluster, based on absorption spectrophotometry, EPR spectroscopy and iron content. Recombinant RnfC contained at least one ironsulfur cluster, most likely of the [4Fe-4S] type. Unambiguous identification of the prosthetic groups was, however, precluded by the extreme instability of this protein. In R. capsulatus, RnfB and RnfC were found by immunoblot analysis to be tightly bound to the membrane, despite their hydrophilic character. The RnfB and RnfC proteins were absent in mutant strains bearing insertions at various positions within the rnfABCDGEH operon, suggesting that their stability depends on the cosynthesis of the other rnf gene products. We observed that iron limitation during growth resulted in a decrease both in the cellular content of RnfB and in the level of transcription of the rnfABCDGEH operon, indicating that the expression of this operon is regulated as a function of iron availability
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.