We have used systematic fluorescence resonance energy transfer and distance-constrained docking to define the three-dimensional structures of bacterial RNA polymerase holoenzyme and the bacterial RNA polymerase-promoter open complex in solution. The structures provide a framework for understanding sigma(70)-(RNA polymerase core), sigma(70)-DNA, and sigma(70)-RNA interactions. The positions of sigma(70) regions 1.2, 2, 3, and 4 are similar in holoenzyme and open complex. In contrast, the position of sigma(70) region 1.1 differs dramatically in holoenzyme and open complex. In holoenzyme, region 1.1 is located within the active-center cleft, apparently serving as a "molecular mimic" of DNA, but, in open complex, region 1.1 is located outside the active center cleft. The approach described here should be applicable to the analysis of other nanometer-scale complexes.

We have used systematic site-specific protein-DNA photocrosslinking to define interactions between bacterial RNA polymerase (RNAP) and promoter DNA in the catalytically competent RNAP-promoter open complex (RPo). We have mapped more than 100 distinct crosslinks between individual segments of RNAP subunits and individual phosphates of promoter DNA. The results provide a comprehensive description of protein-DNA interactions in RPo, permit construction of a detailed model for the structure of RPo, and permit analysis of effects of a transcriptional activator on the structure of RPo.

We report a single-molecule assay that defines, simultaneously, the translocational position of a protein complex relative to DNA and the subunit stoichiometry of the complex. We applied the assay to define translocational positions and sigma70 contents of bacterial transcription elongation complexes in vitro. The results confirm ensemble results indicating that a large fraction, approximately 70%-90%, of early elongation complexes retain sigma70 and that a determinant for sigma70 recognition in the initial transcribed region increases sigma70 retention in early elongation complexes. The results establish that a significant fraction, approximately 50%-60%, of mature elongation complexes retain sigma70 and that a determinant for sigma70 recognition in the initial transcribed region does not appreciably affect sigma70 retention in mature elongation complexes. The results further establish that, in mature elongation complexes that retain sigma70, the half-life of sigma70 retention is long relative to the time-scale of elongation, suggesting that some complexes may retain sigma70 throughout elongation.

The -subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation. This function depends on specific intersubunit interactions that occur when associates with the RNAP core enzyme to form RNAP holoenzyme. Among these interactions, that between conserved region 4 of and the flap domain of the RNAP ␤-subunit (␤-flap) is critical for recognition of the major class of bacterial promoters. Here, we describe the isolation of amino acid substitutions in region 4 of Escherichia coli 70 that have specific effects on the 70 region 4͞␤-flap interaction, either weakening or strengthening it. Using these 70 mutants, we demonstrate that the region 4͞␤-flap interaction also can affect events occurring downstream of transcription initiation during early elongation. Specifically, our results provide support for a structure-based proposal that, when bound to the ␤-flap, region 4 presents a barrier to the extension of the nascent RNA as it emerges from the RNA exit channel. Our findings support the view that the transition from initiation to elongation involves a staged disruption of -core interactions.transcription ͉ promoter escape ͉ region 4 ͉ bacteriophage ͉ PR Ј T he bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (␣ 2 ␤␤Ј) complexed with a factor. factors confer on the holoenzyme the ability to initiate promoter-specific transcription (1). The primary factor in Escherichia coli is 70 , and a typical 70 -dependent promoter bears two conserved sequence elements, the Ϫ10 and the Ϫ35 hexamers, that are separated by a spacer of Ϸ17 bp (1). All primary factors share four regions of conserved sequence (regions 1-4) (2). Regions 2, 3, and 4 contain DNA-binding domains responsible for recognition of the promoter Ϫ10 element, extended Ϫ10 element (3), and Ϫ35 element, respectively (1, 4). Regions 3 and 4 are separated by a flexible linker (region 3.2) (5, 6).70 ordinarily recognizes promoter sequences only in the context of the holoenzyme, formation of which depends critically on a high-affinity interaction between region 2 and a coiled-coil motif in the ␤Ј-subunit (7,8). A weaker interaction between region 4 and the flap domain of the ␤-subunit (␤-flap) is not required for holoenzyme formation, but is required to position region 4 for sequence-specific interaction with the promoter Ϫ35 element (9).During the transition from initiation to elongation, the interaction between and the remainder of the elongation complex is weakened (10-15). Structural evidence has led to the proposal that this is due, at least in part, to sequential displacement of portions of from the core enzyme by the newly synthesized RNA (5, 6, 16). Specifically, structures of RNAP holoenzyme (5, 6, 16) and structural models of the RNAP promoter open complex (16,17) indicate that two regions of lie along the predicted path of the nascent RNA: (i) region 3.2, which is positioned within the RNA exit channel; and (ii) region 4, which, by virtue of its interaction with the ␤-flap, is positioned imme...

Using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic GE23077 (GE) binds directly to the bacterial RNA polymerase (RNAP) active-center ‘i’ and ‘i+1’ nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. The target-based resistance spectrum for GE is unusually small, reflecting the fact that the GE binding site on RNAP includes residues of the RNAP active center that cannot be substituted without loss of RNAP activity. The GE binding site on RNAP is different from the rifamycin binding site. Accordingly, GE and rifamycins do not exhibit cross-resistance, and GE and a rifamycin can bind simultaneously to RNAP. The GE binding site on RNAP is immediately adjacent to the rifamycin binding site. Accordingly, covalent linkage of GE to a rifamycin provides a bipartite inhibitor having very high potency and very low susceptibility to target-based resistance.DOI: http://dx.doi.org/10.7554/eLife.02450.001

CRISPR-Cas9 is widely applied for genome engineering in various organisms. The assembly of single guide RNA (sgRNA) with the Cas9 protein may limit the Cas9/sgRNA effector complex function. We developed a FRET-based assay for detection of CRISPR–Cas9 complex binding to its targets and used this assay to investigate the kinetics of Cas9 assembly with a set of structurally distinct sgRNAs. We find that Cas9 and isolated sgRNAs form the effector complex efficiently and rapidly. Yet, the assembly process is sensitive to the presence of moderate concentrations of non-specific RNA competitors, which considerably delay the Cas9/sgRNA complex formation, while not significantly affecting already formed complexes. This observation suggests that the rate of sgRNA loading into Cas9 in cells can be determined by competition between sgRNA and intracellular RNA molecules for the binding to Cas9. Non-specific RNAs exerted particularly large inhibitory effects on formation of Cas9 complexes with sgRNAs bearing shortened 3′-terminal segments. This result implies that the 3′-terminal segment confers sgRNA the ability to withstand competition from non-specific RNA and at least in part may explain the fact that use of sgRNAs truncated for the 3′-terminal stem loops leads to reduced activity during genomic editing.

Using fluorescence resonance energy transfer, we show that, in the majority of transcription complexes, sigma(70) is not released from RNA polymerase upon transition from initiation to elongation, but, instead, remains associated with RNA polymerase and translocates with RNA polymerase. The results argue against the presumption that there are necessary subunit-composition differences, and corresponding necessary mechanistic differences, in initiation and elongation. The methods of this report should be generalizable to monitor movement of any molecule relative to any nucleic acid.

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) endonuclease cleaves doublestranded DNA sequences specified by guide RNA molecules and flanked by a protospacer adjacent motif (PAM) and is widely used for genome editing in various organisms. The RNA-programmed Cas9 locates the target site by scanning genomic DNA. We sought to elucidate the mechanism of initial DNA interrogation steps that precede the pairing of target DNA with guide RNA. Using fluorometric and biochemical assays, we studied Cas9/guide RNA complexes with model DNA substrates that mimicked early intermediates on the pathway to the final Cas9/guide RNA-DNA complex. The results show that Cas9/guide RNA binding to PAM favors separation of a few PAM-proximal protospacer base pairs allowing initial target interrogation by guide RNA. The duplex destabilization is mediated, in part, by Cas9/guide RNA affinity for unpaired segments of nontarget strand DNA close to PAM. Furthermore, our data indicate that the entry of double-stranded DNA beyond a short threshold distance from PAM into the Cas9/ single-guide RNA (sgRNA) interior is hindered. We suggest that the interactions unfavorable for duplex DNA binding promote DNA bending in the PAM-proximal region during early steps of Cas9/ guide RNA-DNA complex formation, thus additionally destabilizing the protospacer duplex. The mechanism that emerges from our analysis explains how the Cas9/sgRNA complex is able to locate the correct target sequence efficiently while interrogating numerous nontarget sequences associated with correct PAMs.Cas9 | CRISPR | DNA interrogation | protein-DNA interactions | fluorescence spectroscopy P rokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) gene systems acquire fragments of foreign DNA (protospacers) and insert them as spacers into the CRISPR array in the prokaryotic host genome. Upon subsequent encounters, the complex of Cas proteins with CRISPR RNA (crRNA) bearing the spacer sequence binds and cleaves foreign DNA containing the matching sequence (1-3), thus providing the host organism with adaptive hereditable immunity. The DNA endonuclease Cas9 of type II CRISPR-Cas systems is targeted to specific DNA sequences by a 20-base crRNA spacer that binds to the complementary strand of protospacer DNA, displacing the noncomplementary strand to form an R-loop (4, 5). Both binding and cleavage of target DNA by the Cas9/crRNA complex require a short protospacer adjacent motif (PAM) located immediately downstream of the targeted sequence (6). For the most commonly used Streptococcus pyogenes Cas9 (SpCas9), the PAM sequence is 5′-NGG (7). The perfect match between guide RNA and seven to 12 bases of target DNA at the immediate 5′ side of PAM ("seed" region) is the most critical for DNA binding and cleavage, although limited mispairing in the distal bases is tolerated (8). Two RNA molecules (crRNA and a transactivating crRNA (tracrRNA)) required to guide SpCas9 to targets can be replaced with...