Abstract
AAA+ proteins (ATPases associated with various cellular activities) contribute to many cellular processes and typically function as higher order oligomers permitting the coordination of nucleotide hydrolysis for functional output, which leads to substrate remodeling. The precise mechanisms that enable the relay of nucleotide hydrolysis to their specific functional outputs are largely unknown. Here we use PspF, a specialized AAA+ protein required for enhancer-dependent transcription activation in Escherichia coli, as a model system to address this question. We demonstrate that a conserved asparagine is involved in internal organization of the oligomeric ring, regulation of ATPase activity by "trans" factors, and optimizing substrate remodeling. We provide evidence that the spatial relationship between the asparagine residue and the Walker B motif is one key element in the conformational signaling pathway that leads to substrate remodeling. Such functional organization most likely applies to other AAA+ proteins, including Ltag (simian virus 40), Rep40 (Adeno-associated virus-2), and p97 (Mus musculus) in which the asparagine to Walker B motif relationship is conserved.
Highlights
Of their activities and their organization as ring assemblies raises important issues about how they function as molecular machines to engage with, and remodel, their targets
In contrast to 70-dependent transcription, which is constitutively active, 54-dependent transcription requires specific activators that couple ATP hydrolysis to isomerization of the initial transcriptionally inactive closed complex (CC), to a transcriptionally proficient open complex (OC) (10 – 15). 54-dependent transcription activation is functionally analogous to eukaryotic RNAP II, which requires energy derived from ATP hydrolysis provided by TFIIH [16, 17]
Analysis of the different nucleotide-bound structures of PspF1–275 demonstrated that a tight interaction between Walker B residues Glu-108 and Asn-64 occurs in the ATP-bound state, proposed to facilitate the exposure of the GAFTGA motif
Summary
Of their activities and their organization as ring assemblies raises important issues about how they function as molecular machines to engage with, and remodel, their targets. Interactions between PspF1–275 and (E)54, suggesting that We conclude that the defect in the initial transcription rates Asn-64 may be involved either directly or indirectly in the observed with the N64v is not due to a deficiency in promoter nucleotide-dependent exposure of the GAFTGA containing L1 escape but due to a fault in using ATP hydrolysis to drive OC
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