Modeling Transcription Initiation By Bacterial RNA Polymerase

Many promoters contain a curved DNA component essential for high levels of transcription activity. The present work approached the study of these unusual DNA structures in a twofold manner. First, promoter binding by RNA polymerase from Bacillus subtilis and Escherichia coli was examined using a collection of promoters containing DNA curvature. Promoter binding by both RNA polymerases was governed primarily by the nucleotide sequence at the highly conserved $-$10 and $-$35 regions of the promoters. However, the presence of curved DNA, immediately upstream of the $-$35 region of the promoter, predictably increased binding to those promoters which contained curved DNA by the B. subtilis RNA polymerase. Binding by the E. coli RNA polymerase was modestly affected by DNA curvature. The second approach was to characterize the mechanism involved in transcription stimulation by this curve or intrinsically bent DNA. Generally, the proposed mechanisms for transcription initiation include enzyme conformational changes leading to strand separation. Formation of RNA polymerase-promoter complexes that entail significant conformational changes is sensitive to changes in temperature. A goal in the second part of this work was to test if curved DNA influenced a step in transcription initiation that was sensitive to temperature changes. The formation of open promoter complexes was measured using a B. subtilis phage promoter containing curved DNA upstream of the $-$35 region, the Alu156 promoter, and mutants of Alu156 in which the curved DNA sequences were displaced upstream. Open promoter complexes were measured by (1) a run-off transcription assay limited to a single round of initiation and (2) the direct detection of single stranded DNA using potassium permanganate cleavage. Promoters with properly aligned curved DNA formed open promoter complexes at lower temperatures than promoters with misaligned curved DNA. Also, curved DNA enhanced formation of open promoter complexes as measured by actual strand separation. A model describing the effect of curved DNA on open promoter complexes was proposed.

Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.

Base-specific interactions between promoter DNA and Escherichia coli RNA polymerase are regulated by a sigma (sigma) protein during transcription initiation. To map spatial relations between evolutionarily conserved regions of the primary sigma (sigma 70) and each DNA strand along the lacUV5 promoter in the transcriptionally active "open" complex, we have used a cysteine-tethered cutting reagent to cleave DNA strands. The chemical nuclease FeBABE [iron (S)-1-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate] was conjugated to single-cysteine mutants of sigma 70 at sites 132C, 376C, 396C, 422C, 496C, 517C, or 581C. After formation of open promoter complexes between lacUV5 DNA and RNA polymerase holoenzymes carrying conjugated sigma 70 subunits, we observed promoter DNA cleavage spanning at least 60 bases, between positions -48 and +12. The results show that sigma 70 region 2.1, otherwise implicated in core enzyme binding, is proximal to the nontemplate strand of lacUV5 DNA between the -10 promoter element and positions as far downstream of the transcription start site as +12. Conserved region 3.2 of sigma 70 is proximal to the template strand near the +1 transcription start site, and region 3.1 is positioned between the lacUV5-10 and -35 promoter elements. We propose a model for the orientation of sigma 70 and DNA in the open complex.

Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript.

The rate of formation of transcriptionally competent open complexes between Escherichia coli RNA polymerase (RNAP) and the lambda PR promoter is extraordinarily sensitive to the nature and concentration of the electrolyte ions in the solution. The pseudo-first-order time constant of open complex formation tau obsd, determined in excess RNAP at 25 degrees C as a function of NaCl concentration, is proportional to the concentration product [Na+]12 [RNAP]-1. Consequently, tau obsd is far more sensitive to changes in the salt concentration than to changes in the concentration of RNAP. The origin of this effect is the release of the thermodynamic equivalent of 12 monovalent ions in the process of closed complex formation at the lambda PR promoter. In more complex ionic mixtures, ion-specific stoichiometric effects on tau obsd are observed. These are not ionic strength effects but are instead both valence and species specific. Both the association and dissociation rate constants of RNAP at the lambda PR promoter are strongly salt dependent, varying (in NaCl) as [Na+]-12 and [Na+]8, respectively. Consequently, the equilibrium constant characterizing open complex formation at this promoter varies with [Na+]-20. Electrostatic interactions and counterion release are the major contributors to the binding free energy driving open complex formation in a dilute salt solution. Since the in vivo ionic environment of E. coli (and other cells) is highly variable, these large salt effects are almost certainly of physiological significance. Variations in the intracellular concentrations of inorganic and organic ions, including polyamines, must exert both global and also promoter-specific regulatory effects on the initiation of transcription, as well as on numerous other protein-nucleic acid interactions.

Initiation of transcription occurs through a series of steps starting with the binding of RNA polymerase to a promoter DNA and formation of a closed complex. The closed complexes, then isomerize to open complexes. In the open complexes a portion of the promoter DNA is unwound. Using fluorescence spectroscopy, we have investigated in real-time the mechanism of unwinding of promoter DNA during the transition from closed to open complexes of T7 RNA polymerase. We synthesized DNA templates containing the fluorescent base analog 2-aminopurine in place of adenine at specific positions in a T7 RNA polymerase promoter. We located the 2-aminopurine residues in the presumed melting domain of the promoter at -1, -4, and at -6. The fluorescence of 2-aminopurine increases when the DNA goes from a double-stranded form to a single-stranded form. By spectroscopically monitoring the increase in fluorescence of 2-aminopurine in DNA-T7 RNA polymerase complexes, we obtained kinetic and thermodynamic information for DNA unwinding. In the presence of the initiating nucleotide GTP, conformational transitions in the polymerase-promoter complex leading to strand opening were slower than in its absence. The rate of base pair disruption at -1, -6, and at -4 was also slower in the presence of GTP than in its absence. At 37 degrees C, base pair disruption occurred first at -1 followed by -6 and finally at -4. Open complex formation was temperature-sensitive. Temperature effects at -1, -6, and at -4 were consistent with this order of base pair disruption. The apparent activation energies (Ea) for base pair disruption around -1 and -6 were 14 kcal mol-1 and 50 kcal mol-1, respectively, also suggesting this order of base pair disruption. Transcription initiation assays using G-ladder synthesis revealed that initiation rates were almost the same on all three templates containing the modified base. Unlike strand opening, we did not observe lag times for G-ladder synthesis. We suggest that facile base pair disruption at -1 is sufficient for transcription initiation. Based on these data, it is proposed that the polymerase makes contacts at or near -1 and -6 resulting in untwisting of these base pairs thus creating at least two base pair disruption events at -1 and at -6, which are followed by bidirectional propagation to -4.

Papers concerning transcription, and its regulation at promoters, abound in the scientific literature, but reports about initiation, defined as the moment that the first 3′−5′phosphodiester bond of a new transcript is forged, are scarce. In PNAS, Skalenko et al. (1), working with the bacterial multisubunit DNA-dependent RNA polymerase (RNAP), combine high-throughput DNA base-sequencing methods with structural analysis, to provide insights into how RNAP manages the two partner molecules needed to create that first bond, as each new RNA transcript is born. Textbooks paint a deceptively simple picture of transcript initiation: At the majority of bacterial promoters, the RNAP σ (sigma) subunit orchestrates promoter recognition, and then drives the opening of just over one turn of the duplex DNA to form a “transcription bubble,” which is essential for the template strand to be read (2). This is done almost single-handedly by domain 2 of the σ subunit, which makes specific base-dependent interactions with the nontemplate strand of the promoter DNA, and this allows the single-stranded template strand access to the RNAP catalytic site (3⇓–5). The textbook tells us that, then, two nucleoside triphosphates (NTPs) are selected by base pairing with the template strand bases, denoted “TSS” (for transcription start site) and “TSS+1” (one base downstream), and, once in the product and addition sites, the RNAP catalytic activity facilitates phosphodiester bond formation. However, as so often, the reality is not quite so simple, and, over the past decade, Bryce Nickels, Richard Ebright, and their colleagues at Rutgers University have produced a stellar series of papers that, step by step, have unveiled the mysteries of initiation. The first complication arises from inequalities in how RNAP manages the two DNA strands in the transcription bubble (Fig. 1). Prior to initiation, the so-called −10 hexamer element (consensus 5′-TATAAT-3′) in the nontemplate strand … [↵][1]1Email: s.j.w.busby{at}bham.ac.uk. [1]: #xref-corresp-1-1

Transcription initiation at the Escherichia coli galP1 promoter does not depend on specific nucleotide sequences in the -35 region. Footprint analysis of transcriptionally competent complexes between E. coli RNA polymerase and DNA fragments carrying galP1 shows that RNA polymerase protects sequences as far upstream as -55, whereas sequences around the -35 region are exposed. In contrast, with galP1 derivatives carrying -35 region sequences resembling the consensus, RNA polymerase protects bases as far as -45, and the -35 region is fully protected. Taken together, our data suggest that the overall architecture of RNA polymerase-promoter complexes can vary according to whether or not consensus -35 region sequences are present; in the absence of these sequences, open complex formation requires distortion of the promoter DNA. However, the unwinding of promoter DNA around the transcription start is not affected by the nature of the -35 region sequence. With a galP1 derivative carrying point mutations in the spacer region that greatly reduce promoter activity, the protection of bases by RNA polymerase around the -10 sequence and transcription start site is reduced. In contrast, protection of the region upstream of -25 is unaffected by the spacer mutations, although sequences from -46 to -54 become hypersensitive to attack by potassium permanganate, indicating severe distortion or kinking of this zone. We suggest that, with this galP1 derivative, RNA polymerase is blocked in a complex that is an intermediate on the path to open complex formation.

Objectives: To analyze the Multi-Subunit (MSU) DNA dependent RNA polymerases of eubacteria and identify conserved motifs, active site regions among them and propose a plausible mechanism of action for these enzymes, using the E. coli RNA polymerase as a model system. Methods: The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNA polymerases from eubacteria. Along with the conserved motifs identified by the bioinformatics analysis, the data already available from biochemical and Site-Directed Mutagenesis (SDM) experiments and X-ray crystallographic analysis on these enzymes were used to confirm the possible amino acids involved in the active sites and catalysis. Findings: Multiple Sequence Alignment (MSA) of eubacterial RNA polymerases from different bacterial sources showed only a very few highly conserved motifs among them. Possible catalytic regions in the catalytic subunits β and β‘ (in eubacteria) consist of an absolutely conserved amino acid R, in contrast to a K that is reported for DNA polymerases and single subunit (SSU)DNA dependent RNA polymerases. In addition to, the invariant ‘gatekeeper/DNA template binding’ YG pair is also found in all the MSU RNA polymerases as found in all the SSU RNA polymerases and DNA polymerases. The eubacterial βsubunits are very similar in theiractive sites, catalytic regions and distance conservations between the template binding YG pair and the catalytic R, i.e., they are placed at 7 amino acids length. An invariant R is placed here at -6th position from the catalytic R, similar to SSU RNA polymerases and DNA polymerases which is proposed to play an important role in NTP selection. In the eubacterial β’ catalytic subunits, the proposed catalytic R is placed double the distance, i.e., -16 amino acids downstream from the YG pair unlike in the SSU RNA polymerasesand DNA polymerases where the distance is only 8th amino acids downstream from the YG pair. The two Ds in the completely conserved metal binding region could act as the charge shielder and orient the α-phosphates of incoming NTPs for reaction at the 3’-OH growing end. Based on the sequence analysis, a model is proposed for the initiation and elongation steps in the transcription process and a plausible mechanism of action. Applications: A comparative analysis of the bacterial and human RNA polymerases for their transcription mechanism will pave way for design new and effective drugs for many bacterial infections, including the antibiotic resistance. Keywords: Bacterial RNA Polymerases, Clustal Omega, Conserved Motifs, Multi-Subunit RNA Polymerases, Polymerase Active Sites, Polymerization Mechanism, Proofreading

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ70-RNAP and the λPR promoter. Cryo-EM snapshots revealed that the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As the nt-strand 'read-out' extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating that yet unknown conformational changes complete RPo formation in subsequent steps. Given that these events likely describe DNA opening at many bacterial promoters, this study provides insights into how DNA sequence regulates steps of RPo formation.

Transcription initiation by eukaryotic RNA polymerase (Pol) III relies on the TFIIE-related subcomplex C82/34/31. Here we combine cross-linking and hydroxyl radical probing to position the C82/34/31 subcomplex around the Pol III active center cleft. The extended winged helix (WH) domains 1 and 4 of C82 localize to the polymerase domains clamp head and clamp core, respectively, and the two WH domains of C34 span the polymerase cleft from the coiled-coil region of the clamp to the protrusion. The WH domains of C82 and C34 apparently cooperate with other mobile regions flanking the cleft during promoter DNA binding, opening, and loading. Together with published data, our results complete the subunit architecture of Pol III and indicate that all TFIIE-related components of eukaryotic and archaeal transcription systems adopt an evolutionarily conserved location in the upper part of the cleft that supports their functions in open promoter complex formation and stabilization.

Effects of a Single Base-pair Deletion in the Bacteriophage λ PRM Promoter : Repression of PRM by Repressor Bound at OR2 and by RNA Polymerase Bound at PR

The enhancer-binding protein NIFA is required for transcriptional activation of nif promoters by the alternative holoenzyme form of RNA polymerase, which contains the sigma factor sigma 54 (sigma N). NIFA hydrolyzes nucleoside triphosphates to catalyze the isomerization of closed promoter complexes to transcriptionally competent open complexes. The activity of NIFA is antagonized by the regulatory protein NIFL in response to oxygen and fixed nitrogen in vivo. We have investigated the requirement for nucleotides in the formation and stability of open promoter complexes by NIFA and inhibition of its activity by NIFL at the Klebsiella pneumoniae nifH promoter. Open complexes formed by sigma 54-containing RNA polymerase are considerably more stable to heparin challenge in the presence of GTP than in the presence of ATP. This differential stability is most probably a consequence of GTP being the initiating nucleotide at this promoter. Adenosine nucleosides are specifically required for Azotobacter vinelandii NIFL to inhibit open complex formation by native NIFA, and the nucleoside triphosphatase activity of NIFA is strongly inhibited by NIFL under these conditions. We propose a model in which NIFL modulates the activity of NIFA via an adenosine nucleotide switch.

Cycling through transcription with the RNA polymerase F/E (RPB4/7) complex: structure, function and evolution of archaeal RNA polymerase