Insights into the architecture and stoichiometry of Escherichia coli PepA*DNA complexes involved in transcriptional control and site-specific DNA recombination by atomic force microscopy

Abstract

Multifunctional Aminopeptidase A (PepA) from Escherichia coli is involved in the control of two distinct DNA transaction processes: transcriptional repression of the carAB operon, encoding carbamoyl phosphate synthase and site-specific resolution of ColE1-type plasmid multimers. Both processes require communication at a distance along a DNA molecule and PepA is the major structural component of the nucleoprotein complexes that underlie this communication. Atomic Force Microscopy was used to analyze the architecture of PepA·carAB and PepA·cer site complexes. Contour length measurements, bending angle analyses and volume determinations demonstrate that the carP1 operator is foreshortened by ∼235 bp through wrapping around one PepA hexamer. The highly deformed part of the operator extends from slightly upstream of the –35 hexamer of the carP1 promoter to just downstream of the IHF-binding site, and comprises the binding sites for the PurR and RutR transcriptional regulators. This extreme remodeling of the carP1 control region provides a straightforward explanation for the strict requirement of PepA in the establishment of pyrimidine and purine-specific repression of carAB transcription. We further provide a direct physical proof that PepA is able to synapse two cer sites in direct repeat in a large interwrapped nucleoprotein complex, likely comprising two PepA hexamers.

Trypanosoma brucei 20 S Editosomes Have One RNA Substrate-binding Site and Execute RNA Unwinding Activity

“…All of the images were scanned at a frequency of 1 Hz and were analyzed using the MFP-3D software (Asylum Research). Apparent particle volumes (V app ) were calculated as V app ϭ h(w) 2 /6 (where h is height, and w is width), assuming oblate spheroid shapes of the complexes (39). Contour length measurements were performed by manually tracing using MFP-3D and fitting of the contour length histogram to a Gaussian distribution.…”

The functional landscape bound to the transcription factors of Escherichia coli K-12

“…In low proportions, DNAbinding structures, such as IHF-like DNA binding proteins, ribbonhelix-helix and flagellar transcriptional activator FlhD structures were identified. An intriguing observation is the emergence of proteins traditionally not associated with DNA-binding, such as "aminopeptidase A/I" involved in the synthesis of carbamoyl phosphate, an intermediate to the pyrimidine nucleotide pathway (Minh et al, 2009) and RelE-like identified in the regulator RelE associated with toxin-antitoxin systems (Gotfredsen and Gerdes, 1998). In this regard, a hypothetical limit in the number and diversity of DNA-binding TFs can be proposed based on the number of TFs identified and the diversity of structural domains associated with DNA-binding, whereas new DNA-binding structures associated to TFs could emerge in few proportions (Itzkovitz et al, 2006).…”

Hydrolysis of Amides

“…This work also led to the proposal of a new model for the synaptic cer complex in which the two cer sites are wrapped around a single PepA hexamer. In contrast with this, recent studies applying atomic force microscopy to analyze the architecture of this synaptic complex showed that both the shape and the volume of the PepAÁcer complex suggested the presence of two PepA hexamers in the interwrapped synaptic complex instead of one [254]. More research, for instance to determine the stoichiometry of the synaptic complex, is needed to conclusively establish this complexs architecture.…”

“…In this regulatory process, PepA induces an extreme remodeling of one of the two car control regions (carP1), ensuring the cross-talk between various upstream bound transcription regulators (IHF, PyrH, PurR, and RutR) and the RNA polymerase [254]. Similar to its role in the cer/Xer-mediated resolution of ColE1 plasmid multimers, the aminopeptidase activity of E. coli PepA is dispensable for this regulatory role in the expression of the carAB operon [255].…”