RESUMO
The assembly of transcription complexes on eukaryotic promoters involves a series of steps, including chromatin remodeling, recruitment of TATA-binding protein (TBP)-containing complexes, the RNA polymerase II holoenzyme, and additional basal transcription factors. This review describes the transcriptional regulation by TBP and its corresponding homologs that constitute the TBP family and their interactions with promoter DNA. The C-terminal core domain of TBP is highly conserved and contains two structural repeats that fold into a saddle-like structure, essential for the interaction with the TATA-box on DNA. Based on the TBP C-terminal core domain similarity, three TBP-related factors (TRFs) or TBP-like factors (TBPLs) have been discovered in metazoans, TRF1, TBPL1, and TBPL2. TBP is autoregulated, and once bound to DNA, repressors such as Mot1 induce TBP to dissociate, while other factors such as NC2 and the NOT complex convert the active TBP/DNA complex into inactive, negatively regulating TBP. TFIIA antagonizes the TBP repressors but may be effective only in conjunction with the RNA polymerase II holoenzyme recruitment to the promoter by promoter-bound activators. TRF1 has been discovered inDrosophila melanogasterandAnophelesbut found absent in vertebrates and yeast. TBPL1 cannot bind to the TATA-box; instead, TBPL1 prefers binding to TATA-less promoters. However, TBPL1 shows a stronger association with TFIIA than TBP. The TCT core promoter element is present in most ribosomal protein genes inDrosophilaand humans, and TBPL1 is required for the transcription of these genes. TBP directly participates in the DNA repair mechanism, and TBPL1 mediates cell cycle arrest and apoptosis. TBPL2 is closely related to its TBP paralog, showing 95% sequence similarity with the TBP core domain. Like TBP, TBPL2 also binds to the TATA-box and shows interactions with TFIIA, TFIIB, and other basal transcription factors. Despite these advances, much remains to be explored in this family of transcription factors.
Assuntos
RNA Polimerase II , Proteína de Ligação a TATA-Box , Fatores de Transcrição , Transcrição Gênica , Adenosina Trifosfatases/genética , Animais , DNA/genética , Drosophila , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Proteínas Nucleares/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , TATA Box/genética , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/química , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/genética , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Fatores Associados à Proteína de Ligação a TATA , Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/genética , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIIA/genética , Fator de Transcrição TFIIA/metabolismo , Fatores de Transcrição/genéticaRESUMO
The protein kinase A (PKA) signaling pathway plays a role in regulating growth and differentiation in the dimorphic fungus Mucor circinelloides. PKA holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. In M. circinelloides, four genes encode the PKAR1, PKAR2, PKAR3, and PKAR4 isoforms of R subunits. We have constructed null mutants and demonstrate that each isoform has a different role in growth and differentiation. The most striking finding is that pkaR4 is an essential gene, because only heterokaryons were obtained in knockout experiments. Heterokaryons with low levels of wild-type nuclei showed an impediment in the emission of the germ tube, suggesting a pivotal role of this gene in germ tube emergence. The remaining null strains showed different alterations in germ tube emergence, sporulation, and volume of the mother cell. The pkaR2 null mutant showed an accelerated germ tube emission and was the only mutant that germinated under anaerobic conditions when glycine was used as a nitrogen source, suggesting that pkaR2 participates in germ tube emergence by repressing it. From the measurement of the mRNA and protein levels of each isoform in the wild-type and knockout strains, it can be concluded that the expression of each subunit has its own mechanism of differential regulation. The PKAR1 and PKAR2 isoforms are posttranslationally modified by ubiquitylation, suggesting another regulation point in the specificity of the signal transduction. The results indicate that each R isoform has a different role in M. circinelloides physiology, controlling the dimorphism and contributing to the specificity of cyclic AMP (cAMP)-PKA pathway.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Morfogênese , Mucor/enzimologia , Mucor/crescimento & desenvolvimento , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Fúngicas/genética , Deleção de Genes , Holoenzimas/genética , Holoenzimas/metabolismo , Morfogênese/genética , Mucor/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transdução de Sinais , Esporos Fúngicos/crescimento & desenvolvimento , UbiquitinaçãoRESUMO
cAMP-dependent protein kinase (PKA) catalytic (C) and regulatory (R) subunits from Yarrowia lipolytica are encoded by single genes, TPK1 and RKA1, respectively. Here we performed the heterologous expression, purification and characterization of the R subunit from Y. lipolytica yeast cells, and explored the main biochemical features of the PKA. The purified recombinant R, active and capable to interact with C subunit was used to prepare highly specific polyclonal antiserum. Sucrose-gradient centrifugation and gel filtration analysis of both recombinant and native R revealed the monomeric nature of this subunit. Hydrodynamic parameters of the holoenzyme indicated that Y. lipolytica PKA is a dimer of 90 kDa composed of an R subunit of 42 kDa and a C subunit of 39 kDa. The identification of the N-terminal sequence was carried out by mass spectrometry analysis of the purified native R subunit. The differences between N-terminal sequences of R subunits from Y. lipolytica and other organisms, particularly a short linker that spans the inhibitory site, were discussed as the possible cause of the lack of dimerization. R was identified as a type II subunit since our results indicated that it was phosphorylated in vivo by C at S124 identified by anti-phospho-PKA substrate (RRXS/T) antibody.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Yarrowia/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Proteínas Quinases Dependentes de AMP Cíclico/genética , Expressão Gênica , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Hidrodinâmica , Dados de Sequência Molecular , Fosforilação , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Yarrowia/química , Yarrowia/genéticaRESUMO
The specificity in phosphorylation by kinases is determined by the molecular recognition of the peptide target sequence. In Saccharomyces cerevisiae, the protein kinase A (PKA) specificity determinants are less studied than in mammalian PKA. The catalytic turnover numbers of the catalytic subunits isoforms Tpk1 and Tpk2 were determined, and both enzymes are shown to have the same value of 3 s(-1). We analyze the substrate behavior and sequence determinants around the phosphorylation site of three protein substrates, Pyk1, Pyk2, and Nth1. Nth1 protein is a better substrate than Pyk1 protein, and both are phosphorylated by either Tpk1 or Tpk2. Both enzymes also have the same selectivity toward the protein substrates and the peptides derived from them. The three substrates contain one or more Arg-Arg-X-Ser consensus motif, but not all of them are phosphorylated. The determinants for specificity were studied using the peptide arrays. Acidic residues in the position P+1 or in the N-terminal flank are deleterious, and positive residues present beyond P-2 and P-3 favor the catalytic reaction. A bulky hydrophobic residue in position P+1 is not critical. The best substrate has in position P+4 an acidic residue, equivalent to the one in the inhibitory sequence of Bcy1, the yeast regulatory subunit of PKA. The substrate effect in the holoenzyme activation was analyzed, and we demonstrate that peptides and protein substrates sensitized the holoenzyme to activation by cAMP in different degrees, depending on their sequences. The results also suggest that protein substrates are better co-activators than peptide substrates.