Using single-molecule fluorescence resonance energy transfer, we have defined bacterial RNA polymerase (RNAP) clamp formation at each step in transcription initiation and elongation. We find that the clamp predominantly is open in free RNAP and early intermediates in transcription initiation, but closes upon formation of a catalytically competent transcription initiation complex and remains closed during initial transcription and transcription elongation. We show that four RNAP inhibitors interfere with clamp opening. We propose that clamp opening allows DNA to be loaded into and unwound in the RNAP active-center cleft, that DNA loading and unwinding trigger clamp closure, and that clamp closure accounts for the high stability of initiation complexes and the high stability and processivity of elongation complexes.

Transcription by RNA polymerase (RNAP) is often regulated by interactions with control proteins to link specific gene expression to environmental signals and temporal cues. Often activators help recruit RNAP to promoters to increase initiation rates (Busby and Ebright 1999). In contrast, activity of the bacterial 54 containing RNAP holoenzyme is regulated at the DNA melting step (for review, see Buck et al. 2000). Hydrolysis of an NTP by an activator drives a change in configuration of the 54 -holoenzyme, converting the initial closed complex to an open complex to allow interaction with the template DNA for mRNA synthesis (Wedel and Kustu 1995). Preopening of DNA templates does not overcome the requirement for NTP hydrolysis by an activator to promote engagement of the holoenzyme with the melted DNA (Wedel and Kustu 1995;Cannon et al. 1999).The activators of 54 -holoenzyme are members of the large AAA+ protein family, which use ATP binding and hydrolysis to remodel their substrates (Neuwald et al. 1999;Cannon et al. 2000Cannon et al. , 2001. The greater part of the central domain of 54 activators corresponds to the AAA core structure, and includes ATP-binding and hydrolyzing determinants. The 54 protein is known to be the primary target for the NTPase of activators, but how activators use NTP binding and hydrolysis is not well understood (Cannon et al. 2000). Similarly, the nature of the interaction between 54 and the activator is not well described, but an interaction with 54 can be detected in the case of the DctD activator by protein cross-linking (Lee and Hoover 1995). Here we show that the use of ADP-aluminum fluoride, an analog of ATP that mimics ATP in the transition state for hydrolysis, allows formation of a stable complex among the activator PspF, the PspF and NifA central activating domains, and 54 . The binding assay was used to help define determinants in 54 and the activator needed for their interaction, and to show that binding can lead to an altered 54 -DNA footprint. The need for a transition-state analog of ATP for protein-protein binding is discussed in relation to the required ATPase activity of activators of 54 -dependent transcription. In particular, it seems that altered functional states of activators exist as ATP is hydrolyzed. This suggests a parallel to some switch and motor proteins that use nucleotide binding and hydrolysis to establish alternate functional states (Hirose and Amos 1999).

Activators of bacterial σ 54 -RNA polymerase holoenzyme are mechanochemical proteins that use ATP hydrolysis to activate transcription. We have determined a 20 Å resolution structure of an activator, PspF , bound to an ATP transition state analog (ADP.AlF x ), in complex with its basal factor σ 54 by cryo-electron microscopy. By fitting the crystal structure of apo PspF at 1.75 Å into the EM map we identify two loops involved in binding σ 54 . By comparing enhancerbinding structures in different nucleotide states and mutational analysis, we propose nucleotide dependent conformational changes that free the loops for association with σ 54 .Gene expression is regulated at the level of RNA polymerase (RNAP) activity. Bacterial RNAP containing the σ 54 factor requires specialized activator proteins, referred to as bacterial Enhancer-Binding Proteins (EBPs) that interact with the basal transcription complex from remote DNA sites by DNA looping (1-4). EBPs bind Upstream Activating Sequences (UAS) via their C-terminal DNA-binding domains and form higher order oligomers that use ATP-hydrolysis to activate transcription (5, 6). EBPs' central σ 54 -RNAP interacting domain is responsible for ATPase activity and transcription activation (7-9) and belongs to the larger AAA+ (ATPase Associated with various cellular Activities) family of proteins (10-12). Well studied EBPs include Phage Shock protein F (PspF), nitrogen fixation protein A (NifA), nitrogen regulation protein C (NtrC), and C 4 -dicarboxylic acid transport protein D (DctD) (1-3, 7, 13).PspF from Escherichia coli forms a stable oligomeric complex with σ 54 at the point of ATP hydrolysis (14). PspF-ADP.AlF x alters the interaction between σ 54 and promoter DNA similarly to PspF hydrolyzing ATP (15), and was thus deemed a functional hydrolysis intermediate. Activator nucleotide-hydrolysis dependent events couple the chemical energy of hydrolysis to transcriptional activation. The highly conserved and EBP-specific GAFTGA amino acid motif (Fig. S1) is a crucial mechanical determinant for the successful transfer of energy from ATP hydrolysis in EBP to the RNAP holoenzyme via σ 54 's small N-terminal * To whom correspondence should be addressed. xiaodong.zhang@imperial.ac.uk. The lack of structural information has hindered progress towards understanding the basis of this energy transfer process required for transcriptional activation. We now present a structure-function analysis of one such system using: 1) a cryo-electron microscopy reconstruction of PspF's AAA+ domain (residues 1-275, PspF ) in complex with σ 54 at the point of ATP hydrolysis (mimicked by in-situ formed ADP.AlF x ), 2) the crystal structure of apo PspF (1-275) at 1.75 Å resolution, and 3) mutational analysis. Europe PMC Funders GroupNano-electro spray mass spectroscopy of a PspF (1-275) -σ 54 complex with ADP.AlF x established that six monomers of PspF are in complex with a monomeric σ 54 , consistent with AAA+ proteins functioning as hexamers (10, 12).The 3-dimensional reconstruction of the...