RESUMO
In trypanosomes, the production of mRNA relies on the synthesis of the spliced leader (SL) RNA. Expression of the SL RNA is initiated at the only known RNA polymerase II promoter in these parasites. In the pathogenic trypanosome, Trypanosoma brucei, transcription factor IIB (tTFIIB) is essential for SL RNA gene transcription and cell viability, but has a highly divergent primary sequence in comparison to TFIIB in well-studied eukaryotes. Here we describe the 2.3 A resolution structure of the C-terminal domain of tTFIIB (tTFIIB(C)). The tTFIIB(C) structure consists of 2 closely packed helical modules followed by a C-terminal extension of 32 aa. Using the structure as a guide, alanine substitutions of basic residues in regions analogous to functionally important regions of the well-studied eukaryotic TFIIB support conservation of a general mechanism of TFIIB function in eukaryotes. Strikingly, tTFIIB(C) contains additional loops and helices, and, in contrast to the highly basic DNA binding surface of human TFIIB, contains a neutral surface in the corresponding region. These attributes probably mediate trypanosome-specific interactions and have implications for the apparent bidirectional transcription by RNA polymerase II in protein-encoding gene expression in these organisms.
Assuntos
Fator de Transcrição TFIIB/química , Trypanosoma brucei brucei/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , DNA/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína , Eletricidade Estática , Homologia Estrutural de Proteína , Fator de Transcrição TFIIB/isolamento & purificação , Fator de Transcrição TFIIB/metabolismo , Transcrição GênicaRESUMO
Crystal structure of the complex between porcine beta-trypsin and the second domain of the Kazal-type ovomucoid turkey egg white trypsin inhibitor (OMTKY2) has been determined at 1.9A resolution. A peptide fragment from the first domain has been crystallized with the complex. Restrained-refinement of the structure led to an R-factor of 0.19 for the 32206 reflections. OMTKY2 exhibits the canonical Kazal-type fold with a central alpha-helix and a short two-stranded anti-parallel beta-sheet. The carbonyl carbon of the reactive site prefers trigonal geometry. The reactive site loop geometry of the inhibitor is complementary to the surface and charge of the binding site in beta-trypsin.
Assuntos
Ovomucina/química , Inibidores da Tripsina/química , Tripsina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Ovomucina/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Eletricidade Estática , Suínos , Tripsina/metabolismo , Inibidores da Tripsina/metabolismo , Perus , Água/química , Água/metabolismoRESUMO
A database analysis was done to study the role of weak interactions such as CHcdots, three dots, centeredO, CHcdots, three dots, centeredPI(m) and NHcdots, three dots, centeredPI(m) in the thermal stability of proteins. The CHcdots, three dots, centeredO and CHcdots, three dots, centeredPI(m) interactions are more in the case of thermophilic proteins as compared to mesophiles. Amino acid analysis showed that hydrophobic amino acids like Val and Ile, and Cys contribute more to CHcdots, three dots, centeredO hydrogen bonds where as Pro and Gly contribute more to CHcdots, three dots, centeredPI(m) interactions. Though NHcdots, three dots, centeredPI(m) interactions are dominated by Lys and Arg in thermophiles and mesophiles, the Arg contribution is significantly higher in thermophiles. Interestingly, Glycine is a predominant contributor to all the weak interactions. The number of aromatic amino acids in the thermophiles is more and hence a large number of aromatic clusters were observed in this class. Thus, a cumulative effect of weak interactions seems to be important in thermal stability of proteins. The study also shows that introduction of Gly, Arg, Phe, Pro, and Tyr may enhance the thermal stability.