The von Hippel-Lindau (VHL) tumor suppressor gene is mutated in most human kidney cancers. The VHL protein is part of a complex that includes Elongin B, Elongin C, and Cullin-2, proteins associated with transcriptional elongation and ubiquitination. Here it is shown that the endogenous VHL complex in rat liver also includes Rbx1, an evolutionarily conserved protein that contains a RING-H2 fingerlike motif and that interacts with Cullins. The yeast homolog of Rbx1 is a subunit and potent activator of the Cdc53-containing SCFCdc4 ubiquitin ligase required for ubiquitination of the cyclin-dependent kinase inhibitor Sic1 and for the G1 to S cell cycle transition. These findings provide a further link between VHL and the cellular ubiquitination machinery.
Background: Surfactant protein D (SP-D) plays an important role in innate defense against influenza A viruses (IAVs) and other pathogens.
Pompe's disease is caused by a deficiency of the lysosomal enzyme acid ␣-glucosidase (GAA). GAA is synthesized as a 110-kDa precursor containing N-linked carbohydrates modified with mannose 6-phosphate groups. Following trafficking to the lysosome, presumably via the mannose 6-phosphate receptor, the 110-kDa precursor undergoes a series of complex proteolytic and Nglycan processing events, yielding major species of 76 and 70 kDa. During a detailed characterization of human placental and recombinant human GAA, we found that the peptides released during proteolytic processing remained tightly associated with the major species. The 76-kDa form (amino acids (aa) 122-782) of GAA is associated with peptides of 3.9 kDa (aa 78 -113) and 19.4 kDa (aa 792-952). The 70-kDa form (aa 204 -782) contains the 3.9-and 19.4-kDa peptide species as well as a 10.3-kDa species (aa 122-199). A similar set of proteolytic fragments has been identified in hamster GAA, suggesting that the multicomponent character is a general phenomenon. Rabbit anti-peptide antibodies have been generated against sequences in the proteolytic fragments and used to demonstrate the time course of uptake and processing of the recombinant GAA precursor in Pompe's disease fibroblasts. The results indicate that the observed fragments are produced intracellularly in the lysosome and not as a result of nonspecific proteolysis during purification. These data demonstrate that the mature forms of GAA characterized by polypeptides of 76 or 70 kDa are in fact larger molecular mass multicomponent enzyme complexes.Lysosomal acid ␣-glucosidase (GAA 1 ; EC 3.2.1.3) is an exo-1,4-and -1,6-␣-glucosidase that hydrolyzes glycogen to glucose. The cDNA for GAA encodes a protein of 952 amino acids with a predicted molecular mass of 105 kDa (1). The newly synthesized precursor has an amino-terminal signal peptide for cotranslational transport into the lumen of the endoplasmic reticulum, where it is N-glycosylated at seven glycosylation sites, resulting in a glycosylated precursor with an apparent molecular mass of 110 kDa.The intracellular processing of GAA has been investigated previously (2, 3). It was proposed that, after transport through the Golgi complex and targeting to the endosome/lysosome, the 110-kDa precursor is proteolytically processed at the amino terminus, resulting in a 95-kDa intermediate with a sequence beginning at amino acid 122. Prior to this study, the 95-kDa intermediate was proposed to be proteolytically processed to a 76-kDa form, which was believed to occur between amino acids 816 and 881 (3). The 76-kDa form is then proteolytically processed at the amino terminus at amino acid 204 to give the 70-kDa mature form (3). The nomenclature used for the processed forms of GAA is based on apparent molecular mass as determined by SDS-PAGE.The identities of the proteases involved in the maturation of GAA have never been established. GAA has been purified from many different tissues such as bovine testis (4), rat liver (5), pig liver (6), human liver (7), rabbit mus...
TFIIH is an RNA polymerase II transcription factor that performs ATP-dependent functions in both transcription initiation, where it catalyzes formation of the open complex, and in promoter escape, where it suppresses arrest of the early elongation complex at promoter-proximal sites. TFIIH possesses three known ATPdependent activities: a 3 3 5 DNA helicase catalyzed by its XPB subunit, a 5 3 3 DNA helicase catalyzed by its XPD subunit, and a carboxyl-terminal domain (CTD) kinase activity catalyzed by its CDK7 subunit. In this report, we exploit TFIIH mutants to investigate the contributions of TFIIH DNA helicase and CTD kinase activities to efficient promoter escape by RNA polymerase II in a minimal transcription system reconstituted with purified polymerase and general initiation factors. Our findings argue that the TFIIH XPB DNA helicase is primarily responsible for preventing premature arrest of early elongation intermediates during exit of polymerase from the promoter.TFIIH is a nine-subunit complex that possesses multiple catalytic activities, including DNA-dependent ATPase, DNA helicase, and a protein kinase that is capable of phosphorylating the carboxyl-terminal domain (CTD) 1 of the largest subunit of RNA polymerase II (1). The two largest TFIIH subunits are ATP-dependent DNA helicases encoded by the Xeroderma pigmentosum complementation group B (XPB) and D (XPD) genes. The TFIIH-associated CTD kinase is a three-subunit subassembly, CDK-activating kinase (CAK), which is composed of the kinase/cyclin pair CDK7/cyclin H and the RING-H2 finger protein MAT1. TFIIH subunits are found in a variety of additional subassemblies, including a six-subunit complex (IIH6) containing XPB, XPD, p62, p52, p44, and p34, a five-subunit "core" complex (IIH5) containing XPB, p62, p52, p44, and p34, and a four-subunit XPD/CAK complex (2-6).TFIIH was initially identified by its requirement in transcription initiation by RNA polymerase II (7). Initiation is an ATP-dependent process that requires at minimum the five general initiation factors TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (8,9). Biochemical studies have shown that initiation in this minimal transcription system proceeds through multiple stages beginning with assembly of polymerase and all five general initiation factors into a closed preinitiation complex at the promoter (8, 9) and culminating in ATP-dependent formation of the open complex and synthesis of the first phosphodiester bond of nascent transcripts (10 -13). Evidence supporting a role for TFIIH DNA helicase activity in ATP-dependent formation of the open complex was initially suggested by studies indicating that both TFIIH and ATP are dispensible for initiation by RNA polymerase II from artificial promoters containing premelted transcriptional start sites and from promoters on negatively supercoiled DNA templates (14 -19).In addition to its requirement in transcription initiation, TFIIH is also required for efficient promoter escape by RNA polymerase II (18, 20 -22). Mechanistic studies have shown that a fra...
Transcription factor (TF) IIF is a multifunctional RNA polymerase II transcription factor that has well established roles in both transcription initiation, where it functions as a component of the preinitiation complex and is required for formation of the open complex and synthesis of the first phosphodiester bond of nascent transcripts, and in transcription elongation, where it is capable of interacting directly with the ternary elongation complex and stimulating the rate of transcription. In this report, we present evidence that TFIIF is also required for efficient promoter escape by RNA polymerase II. Our findings argue that TFIIF performs dual roles in this process. We observe (i) that TFIIF suppresses the frequency of abortive transcription by very early RNA polymerase II elongation intermediates by increasing their processivity and (ii) that TFIIF cooperates with TFIIH to prevent premature arrest of early elongation intermediates. In addition, our findings argue that two TFIIF functional domains mediate TFIIF action in promoter escape. First, we observe that a TFIIF mutant selectively lacking elongation activity supports TFIIH action in promoter escape, but is defective in suppressing the frequency of abortive transcription by very early RNA polymerase II elongation intermediates. Second, a TFIIF mutant selectively lacking initiation activity is more active than wild type TFIIF in increasing the processivity of very early elongation intermediates, but is defective in supporting TFIIH action in promoter escape. Taken together, our findings bring to light a function for TFIIF in promoter escape and support a role for TFIIF elongation activity in this process. TFIIF1 was originally identified by its requirement in promoter-specific transcription initiation by RNA polymerase II. Mammalian TFIIF is a heterodimer composed of ϳ30 kDa (RAP30) and ϳ74 kDa (RAP74) subunits (1). Substantial evidence suggests that TFIIF performs multiple functions in transcription initiation. Although TFIIF is not essential for selective binding of RNA polymerase II to promoters, TFIIF functions as an integral component of the preinitiation complex and strongly stabilizes binding of polymerase to TFIID and TFIIB at the promoter (2-4). In addition, TFIIF is required for entry of TFIIE and TFIIH into the preinitiation complex (2, 4 -6), for subsequent open complex formation catalyzed by the TFIIH DNA helicase, and for synthesis of the first phosphodiester bond of nascent transcripts (7-9). Although it is presently not known whether TFIIF merely functions as a scaffold for binding of TFIIH to the preinitiation complex or whether TFIIF actively participates in formation of the open complex, evidence suggests that TFIIF interacts with promoter DNA in the preinitiation complex and induces a dramatic conformational change that results in wrapping of DNA for nearly a full turn around RNA polymerase II and may facilitate formation of the open complex (10 -12).In addition to its role in transcription initiation, TFIIF is also capable of potentl...
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