RNAP-promoter Open Complex Research Articles

Structural basis of λCII-dependent transcription activation

The CII protein of bacteriophage λ activates transcription from the phage promoters PRE, PI, and PAQ by binding to two direct repeats that straddle the promoter -35 element. Although genetic, biochemical, and structural studies have elucidated many aspects of λCII-mediated transcription activation, no precise structure of the transcription machinery in the process is available. Here, we report a 3.1-Å cryo-electron microscopy (cryo-EM) structure of an intact λCII-dependent transcription activation complex (TAC-λCII), which comprises λCII, E.coli RNAP-σ70 holoenzyme, and the phage promoter PRE. The structure reveals the interactions between λCII and the direct repeats responsible for promoter specificity and the interactions between λCII and RNAP α subunit C-terminal domain responsible for transcription activation. We also determined a 3.4-Å cryo-EM structure of an RNAP-promoter open complex (RPo-PRE) from the same dataset. Structural comparison between TAC-λCII and RPo-PRE provides new insights into λCII-dependent transcription activation.

Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on aconsensus promoter.

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor n}{}sigma to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein–DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RNAP-promoter open (RPO) complex is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting with n}{}sigma and the RNAP n}{}beta subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.

Molecular dissection of RbpA-mediated regulation of fidaxomicin sensitivity in mycobacteria

RNA polymerase (RNAP) binding protein A (RbpA) is essential for mycobacterial viability and regulates transcription initiation by increasing the stability of the RNAP-promoter open complex (RPo). RbpA consists of four domains: an N-terminal tail (NTT), a core domain (CD), a basic linker, and a sigma interaction domain. We have previously shown that truncation of the RbpA NTT and CD increases RPo stabilization by RbpA, implying that these domains inhibit this activity of RbpA. Previously published structural studies showed that the NTT and CD are positioned near multiple RNAP-σA holoenzyme functional domains and predict that the RbpA NTT contributes specific amino acids to the binding site of the antibiotic fidaxomicin (Fdx), which inhibits the formation of the RPo complex. Furthermore, deletion of the NTT results in decreased Mycobacterium smegmatis sensitivity to Fdx, but whether this is caused by a loss in Fdx binding is unknown. We generated a panel of rbpA mutants and found that the RbpA NTT residues predicted to directly interact with Fdx are partially responsible for RbpA-dependent Fdx activity in vitro, while multiple additional RbpA domains contribute to Fdx activity in vivo. Specifically, our results suggest that the RPo-stabilizing activity of RbpA decreases Fdx activity in vivo. In support of the association between RPo stability and Fdx activity, we find that another factor that promotes RPo stability in bacteria, CarD, also impacts to Fdx sensitivity. Our findings highlight how RbpA and other factors may influence RNAP dynamics to affect Fdx sensitivity.

Clp protease and antisense RNA jointly regulate the global regulator CarD to mediate mycobacterial starvation response

Under starvation conditions, bacteria tend to slow down their translation rate by reducing rRNA synthesis, but the way they accomplish that may vary in different bacteria. In Mycobacterium species, transcription of rRNA is activated by the RNA polymerase (RNAP) accessory transcription factor CarD, which interacts directly with RNAP to stabilize the RNAP-promoter open complex formed on rRNA genes. The functions of CarD have been extensively studied, but the mechanisms that control its expression remain obscure. Here, we report that the level of CarD was tightly regulated when mycobacterial cells switched from nutrient-rich to nutrient-deprived conditions. At the translational level, an antisense RNA of carD (AscarD) was induced in a SigF-dependent manner to bind with carD mRNA and inhibit CarD translation, while at the post-translational level, the residual intracellular CarD was quickly degraded by the Clp protease. AscarD thus worked synergistically with Clp protease to decrease the CarD level to help mycobacterial cells cope with the nutritional stress. Altogether, our work elucidates the regulation mode of CarD and delineates a new mechanism for the mycobacterial starvation response, which is important for the adaptation and persistence of mycobacterial pathogens in the host environment.

Transcription activation by a sliding clamp

Transcription activation of bacteriophage T4 late genes is accomplished by a transcription activation complex containing RNA polymerase (RNAP), the promoter specificity factor gp55, the coactivator gp33, and a universal component of cellular DNA replication, the sliding clamp gp45. Although genetic and biochemical studies have elucidated many aspects of T4 late gene transcription, no precise structure of the transcription machinery in the process is available. Here, we report the cryo-EM structures of a gp55-dependent RNAP-promoter open complex and an intact gp45-dependent transcription activation complex. The structures reveal the interactions between gp55 and the promoter DNA that mediate the recognition of T4 late promoters. In addition to the σR2 homology domain, gp55 has a helix-loop-helix motif that chaperons the template-strand single-stranded DNA of the transcription bubble. Gp33 contacts both RNAP and the upstream double-stranded DNA. Gp45 encircles the DNA and tethers RNAP to it, supporting the idea that gp45 switches the promoter search from three-dimensional diffusion mode to one-dimensional scanning mode.

Structural basis for transcription inhibition by E. coli SspA.

Stringent starvation protein A (SspA) is an RNA polymerase (RNAP)-associated protein involved in nucleotide metabolism, acid tolerance and virulence of bacteria. Despite extensive biochemical and genetic analyses, the precise regulatory role of SspA in transcription is still unknown, in part, because of a lack of structural information for bacterial RNAP in complex with SspA. Here, we report a 3.68 Å cryo-EM structure of an Escherichia coli RNAP-promoter open complex (RPo) with SspA. Unexpectedly, the structure reveals that SspA binds to the E. coli σ70-RNAP holoenzyme as a homodimer, interacting with σ70 region 4 and the zinc binding domain of EcoRNAP β′ subunit simultaneously. Results from fluorescent polarization assays indicate the specific interactions between SspA and σ70 region 4 confer its σ selectivity, thereby avoiding its interactions with σs or other alternative σ factors. In addition, results from in vitro transcription assays verify that SspA inhibits transcription probably through suppressing promoter escape. Together, the results here provide a foundation for understanding the unique physiological function of SspA in transcription regulation in bacteria.

Effects of Discriminator Changes on Open Complex Formation, Stabilization, and Transcription Initiation

The discriminator is the region of promoter DNA between the key −10 recognition region for RNA polymerase (RNAP) and the transcription start site (TSS). The length, sequences of the discriminator, and the interactions between RNAP mobile elements and promoter DNA the discriminator direct, are major determinants of the lifetime and the structure of the RNAP-promoter open complex (OC). The OC lifetime and rate of formation can both vary by ∼4 orders of magnitude for different promoter sequences, and together regulate the rate of initiation and escape of RNAP from these promoters. We are investigating a series of discriminators including those of the long-lived λPR OC (GGTTGC) and the short-lived rrnBP1 OC (GCGCCACC), in the context of the upstream λPR promoter and a modified λPR transcribed region. Fast kinetic methods with filter binding detection are used to determine overall rate constants for open complex formation and dissociation, and to dissect contributions to these rate constants from initial equilibria involving closed or open complexes (CC or OC), and the rate-determining isomerizations step between them. Our results to date indicate that the discriminator affects primarily the steps that stabilize the initial OC, and has only limited effects on the DNA closing rate constant or on the overall second order rate constant for OC formation. From previous studies, interactions of in-cleft (ss) DNA and downstream (ds) DNA with RNAP change in the process of stabilization of the initial OC. Regions 1.1 and 1.2 of σ70 and downstream mobile elements of core RNAP are involved, and the orientation of the downstream duplex is changed. We are investigating these interactions and conformational changes for different discriminators in more detail, and the relationship to initiation and promoter escape of RNAP.

Characterizing highly dynamic conformational states: The transcription bubble in RNAP-promoter open complex as an example.

Bio-macromolecules carry out complicated functions through structural changes. To understand their mechanism of action, the structure of each step has to be characterized. While classical structural biology techniques allow the characterization of a few "structural snapshots" along the enzymatic cycle (usually of stable conformations), they do not cover all (and often fast interconverting) structures in the ensemble, where each may play an important functional role. Recently, several groups have demonstrated that structures of different conformations in solution could be solved by measuring multiple distances between different pairs of residues using single-molecule Förster resonance energy transfer (smFRET) and using them as constrains for hybrid/integrative structural modeling. However, this approach is limited in cases where the conformational dynamics is faster than the technique's temporal resolution. In this study, we combine existing tools that elucidate sub-millisecond conformational dynamics together with hybrid/integrative structural modeling to study the conformational states of the transcription bubble in the bacterial RNA polymerase-promoter open complex (RPo). We measured microsecond alternating laser excitation-smFRET of differently labeled lacCONS promoter dsDNA constructs. We used a combination of burst variance analysis, photon-by-photon hidden Markov modeling, and the FRET-restrained positioning and screening approach to identify two conformational states for RPo. The experimentally derived distances of one conformational state match the known crystal structure of bacterial RPo. The experimentally derived distances of the other conformational state have characteristics of a scrunched RPo. These findings support the hypothesis that sub-millisecond dynamics in the transcription bubble are responsible for transcription start site selection.

The mechanism of variability in transcription start site selection.

During transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing a single-stranded 'transcription bubble,' and selects a transcription start site (TSS). TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. Variability in TSS selection has been proposed to involve DNA 'scrunching' and 'anti-scrunching,' the hallmarks of which are: (i) forward and reverse movement of the RNAP leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the transcription bubble. Here, using in vitro and in vivo protein-DNA photocrosslinking and single-molecule nanomanipulation, we show bacterial TSS selection exhibits both hallmarks of scrunching and anti-scrunching, and we define energetics of scrunching and anti-scrunching. The results establish the mechanism of TSS selection by bacterial RNAP and suggest a general mechanism for TSS selection by bacterial, archaeal, and eukaryotic RNAP.

Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes.

Transcription initiation is a major step in gene regulation for all organisms. In bacteria, the promoter DNA is first recognized by RNA polymerase (RNAP) to yield an initial closed complex. This complex subsequently undergoes conformational changes resulting in DNA strand separation to form a transcription bubble and an RNAP-promoter open complex; however, the series and sequence of conformational changes, and the factors that influence them are unclear. To address the conformational landscape and transitions in transcription initiation, we applied single-molecule Förster resonance energy transfer (smFRET) on immobilized Escherichia coli transcription open complexes. Our results revealed the existence of two stable states within RNAP–DNA complexes in which the promoter DNA appears to adopt closed and partially open conformations, and we observed large-scale transitions in which the transcription bubble fluctuated between open and closed states; these transitions, which occur roughly on the 0.1 s timescale, are distinct from the millisecond-timescale dynamics previously observed within diffusing open complexes. Mutational studies indicated that the σ70 region 3.2 of the RNAP significantly affected the bubble dynamics. Our results have implications for many steps of transcription initiation, and support a bend-load-open model for the sequence of transitions leading to bubble opening during open complex formation.

Flexibility in Transcription Start-Site Selection by RNA Polymerase Involves Transcription-Bubble Expansion (“Scrunching”) or Contraction (“Unscrunching”)

RNA polymerase (RNAP) is a molecular machine that carries out a series of reactions during transcription initiation:(i) RNAP binds to promoter DNA, yielding an RNAP-promoter closed complex (RPc).(ii) RNAP unwinds ∼13 base pairs of promoter DNA surrounding the transcription start site, forming a single-stranded region (transcription bubble), and yielding an RNAP-promoter open complex (RPo).(iii) RNAP begins synthesis of an RNA product as an RNAP-promoter initial transcribing complex (RPitc).(iv) After RNAP synthesizes an RNA product ∼11 nt in length, RNAP breaks its interactions with the promoter, escapes from the promoter, and begins transcription elongation as an RNAP-DNA elongation complex (RDe).It has been known for four decades that the transcription start site can vary over a range of at least 5 bp--comprising the default start site (position +1), downstream-shifted start sites, (positions +2 and +3), and upstream-sifted start sites (positions −2 and −1)--and that the transcription start site can be re-programmed within this range by the use of appropriate ribodinucleotide primers. However, the mechanistic basis of this flexibility in transcription start-site selection has not been known.In this work, we have used magnetic-tweezers single-molecule nanomanipulation to monitor the extent of RNAP-dependent DNA unwinding in transcription initiation complexes containing ribodinucleotide primers that re-program transcription to start at downstream-shifted start sites (positions +2 or +3) or upstream-shifted start sites (positions −2 or −1). The results indicate that re-programming the transcription start site changes the transcription-bubble size: forcing a downstream-shifted start site increases transcription-bubble size, and forcing upstream-shifted start sites decreases transcription-bubble size. The results support a model in which flexibility in transcription start-site selection is a consequence of pre-initiation transcription-bubble expansion (“pre-initiation scrunching”) or pre-initiation transcription-bubble contraction (“pre-initiation unscrunching”).

Structural Basis of Transcription Initiation

During transcription initiation, RNA polymerase (RNAP) binds and unwinds promoter DNA to form an RNAP-promoter open complex. We have determined crystal structures at 2.9 and 3.0 Å resolution of functional transcription initiation complexes comprising Thermus thermophilus RNA polymerase, σ(A), and a promoter DNA fragment corresponding to the transcription bubble and downstream double-stranded DNA of the RNAP-promoter open complex. The structures show that σ recognizes the -10 element and discriminator element through interactions that include the unstacking and insertion into pockets of three DNA bases and that RNAP recognizes the -4/+2 region through interactions that include the unstacking and insertion into a pocket of the +2 base. The structures further show that interactions between σ and template-strand single-stranded DNA (ssDNA) preorganize template-strand ssDNA to engage the RNAP active center.

Upstream promoter sequences and αCTD mediate stable DNA wrapping within the RNA polymerase–promoter open complex

We show that the extent of stable DNA wrapping by Escherichia coli RNA polymerase (RNAP) in the RNAP-promoter open complex depends on the sequence of the promoter and, in particular, on the sequence of the upstream region of the promoter. We further show that the extent of stable DNA wrapping depends on the presence of the RNAP alpha-subunit carboxy-terminal domain and on the presence and length of the RNAP alpha-subunit interdomain linker. Our results indicate that the extensive stable DNA wrapping observed previously in the RNAP-promoter open complex at the lambda P(R) promoter is not a general feature of RNAP-promoter open complexes.

Structural Organization of the RNA Polymerase-Promoter Open Complex

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.