Assuntos
Proteínas de Bactérias/química , Endopeptidase Clp/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Cinética , Mutação , Staphylococcus aureus/enzimologia , Especificidade por SubstratoRESUMO
The eukaryotic RNA polymerases Pol I, II, and III use different promoters to transcribe different classes of genes. Promoter usage relies on initiation factors, including TFIIF and TFIIE, in the case of Pol II. Here, we show that the Pol I-specific subunits A49 and A34.5 form a subcomplex that binds DNA and is related to TFIIF and TFIIE. The N-terminal regions of A49 and A34.5 form a dimerization module that stimulates polymerase-intrinsic RNA cleavage and has a fold that resembles the TFIIF core. The C-terminal region of A49 forms a "tandem winged helix" (tWH) domain that binds DNA with a preference for the upstream promoter nontemplate strand and is predicted in TFIIE. Similar domains are predicted in Pol III-specific subunits. Thus, Pol I/III subunits that have no counterparts in Pol II are evolutionarily related to Pol II initiation factors and may have evolved to mediate promoter specificity and transcription processivity.
Assuntos
Candida glabrata/enzimologia , DNA/metabolismo , RNA Polimerase I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição TFII/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Candida glabrata/genética , Cristalografia por Raios X , DNA/química , Evolução Molecular , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Subunidades Proteicas , RNA Polimerase I/química , RNA Polimerase I/genética , Proteínas Recombinantes/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relação Estrutura-Atividade , Fatores de Transcrição TFII/químicaRESUMO
The removal of flexible protein regions is generally used to promote crystallization, but advanced strategies to quickly remove multiple flexible regions from proteins or protein complexes are lacking. Here, it is shown how a protein heterodimer with multiple flexibilities, the RNA polymerase I subcomplex A14/A43, could be crystallized with the use of an iterative procedure of predicting flexible regions, experimentally testing and improving these predictions and combining deletions of flexible regions in a stepwise manner. This strategy should enable the crystallization of other proteins and subcomplexes with multiple flexibilities, as required for hybrid structure solution of large macromolecular assemblies.
Assuntos
Cristalização/métodos , Engenharia de Proteínas , RNA Polimerase I/química , Sequência de Aminoácidos , Clonagem Molecular , Biologia Computacional , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Polimerase I/metabolismo , RNA Polimerase I/fisiologia , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-A three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite.
Assuntos
Fasciola hepatica/enzimologia , Proteínas de Helminto/química , Modelos Moleculares , Fatores de Virulência/química , Animais , Sítios de Ligação/fisiologia , Catepsinas , Cristalografia por Raios X , Fasciola hepatica/genética , Fasciola hepatica/patogenicidade , Proteínas de Helminto/genética , Humanos , Concentração de Íons de Hidrogênio , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato/fisiologia , Fatores de Virulência/genéticaRESUMO
Synthesis of ribosomal RNA (rRNA) by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic cell growth. Here we report the 12 A cryo-electron microscopic structure for the complete 14-subunit yeast Pol I, a homology model for the core enzyme, and the crystal structure of the subcomplex A14/43. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3'-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2 and, apparently, enables ribosomal RNA proofreading and 3'-end trimming.
