RESUMO
The GTPases Toc159 and Toc34 of the translocon of the outer envelope of chloroplasts (TOC) are involved in recognition and transfer of precursor proteins at the cytosolic face of the organelle. Both proteins engage multiple interactions within the translocon during the translocation process, including dimeric states of their G-domains. The units of the Toc34 homodimer are involved in the recognition of the transit peptide representing the translocation signal of precursor proteins. This substrate recognition is part of the regulation of the GTPase cycle of Toc34. The Toc159 monomer and the Toc34 homodimer recognize the transit peptide of the small subunit of Rubisco at the N- and at the C-terminal region, respectively. Analysis of the transit peptide interaction by crosslinking shows that the heterodimer between both G-domains binds pSSU most efficiently. While substrate recognition by Toc34 homodimer was shown to regulate nucleotide exchange, we provide evidence that the high activation energy of the GTPase Toc159 is lowered by substrate recognition. The nucleotide affinity of Toc34G homodimer and Toc159G monomer are distinct, Toc34G homodimer recognizes GDP and Toc159G GTP with highest affinity. Moreover, the analysis of the nucleotide association rates of the monomeric and dimeric receptor units suggests that the heterodimer has an arrangement distinct from the homodimer of Toc34. Based on the biochemical parameters determined we propose a model for the order of events at the cytosolic side of TOC. The molecular processes described by this hypothesis range from transit peptide recognition to perception of the substrate by the translocation channel.
Assuntos
GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Pisum sativum/enzimologia , Sítios de Ligação , Cloroplastos/enzimologia , GTP Fosfo-Hidrolases/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Multimerização ProteicaRESUMO
A continuous FeMo cofactor supply for nitrogenase maturation is ensured in Azotobacter vinelandii by developing a cage-like molybdenum storage protein (MoSto) capable to store ca. 120 molybdate molecules ( MoO 4 2 - ) as discrete polyoxometalate (POM) clusters. To gain mechanistic insight into this process, MoSto was characterized by Mo and ATP/ADP content, structural, and kinetic analysis. We defined three functionally relevant states specified by the presence of both ATP/ADP and POM clusters (MoStofunct ), of only ATP/ADP (MoStobasal ) and of neither ATP/ADP nor POM clusters (MoStozero ), respectively. POM clusters are only produced when ATP is hydrolyzed to ADP and phosphate. Vmax was ca. 13 µmolphosphate ·min-1 ·mg-1 and Km for molybdate and ATP/Mg2+ in the low micromolar range. ATP hydrolysis presumably proceeds at subunit α, inferred from a highly occupied α-ATP/Mg2+ and a weaker occupied ß-ATP/no Mg2+ -binding site found in the MoStofunct structure. Several findings indicate that POM cluster storage is separated into a rapid ATP hydrolysis-dependent molybdate transport across the protein cage wall and a slow molybdate assembly induced by combined auto-catalytic and protein-driven processes. The cage interior, the location of the POM cluster depot, is locked in all three states and thus not rapidly accessible for molybdate from the outside. Based on Vmax , the entire Mo storage process should be completed in less than 10 s but requires, according to the molybdate content analysis, ca. 15 min. Long-time incubation of MoStobasal with nonphysiological high molybdate amounts implicates an equilibrium in and outside the cage and POM cluster self-formation without ATP hydrolysis. DATABASES: The crystal structures MoSto in the MoSto-F6, MoSto-F7, MoStobasal , MoStozero , and MoSto-F1vitro states were deposited to PDB under the accession numbers PDB 6GU5, 6GUJ, 6GWB, 6GWV, and 6GX4.
Assuntos
Trifosfato de Adenosina/metabolismo , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/metabolismo , Metaloproteínas/metabolismo , Molibdênio/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , Cristalografia por Raios X , Metaloproteínas/química , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
RNA aptamers have been widely used as regulators for conditional gene expression. The TetR binding aptamer can activate tetracycline repressor TetR controlled gene expression with high efficiency. Here we demonstrate that the aptamer can also activate TetR controlled gene expression when expressed in the context of a natural transcripts. The aptamer was inserted into the untranslated regions of mRNAs as well as into small non-coding RNAs and was expressed both from a plasmid and from an endogenous locus. Our data suggest that the aptamer is a valuable tool to easily monitor the expression level of different RNAs, and it therefore represents a powerful tool for the construction of complex synthetic gene networks.
Assuntos
Aptâmeros de Nucleotídeos/química , RNA não Traduzido/biossíntese , RNA/biossíntese , Tetraciclina/metabolismo , Fatores de Transcrição/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Escherichia coli/genética , Expressão Gênica , Dados de Sequência Molecular , Plasmídeos/genética , Ligação Proteica , RNA/análise , RNA/genética , RNA não Traduzido/análise , RNA não Traduzido/genética , Tetraciclina/farmacologia , Transcrição GênicaRESUMO
The investigation of cellular processes on the molecular level is important to understand the functional network within plant cells. self-assembling GFP has evolved to be a versatile tool for (membrane) protein analyses. Based on the autocatalytical reassembling property of the nonfluorescent strands 1-10 and 11, protein distribution and membrane protein topology can be analyzed in vivo. Here, we provide basic protocols to determine membrane protein topology in Arabidopsis thaliana protoplasts.
