Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy.

Bacterial translation initiation entails the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex (IC) to form a 70S IC that subsequently matures into a 70S elongation-competent complex (70S EC). Rapid and accurate 70S IC formation is promoted by 30S IC-bound initiation factors (IFs), which must dissociate before the resulting 70S EC can begin translation elongation1. Although comparison of 30S2–5 and 70S4,6–8 IC structures have revealed that the ribosome, IFs, and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during 70S IC formation, structures of any intermediates formed during these rearrangements, and contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, the absence of a 70S EC structure obtained directly from a 70S IC formed via an IF-catalyzed initiation reaction has precluded an understanding of ribosome, IF, and fMet-tRNAfMet rearrangements that occur upon maturation of a 70S IC into a 70S EC. Using time-resolved cryogenic electron microscopy (TR cryo-EM)9 we report the first, near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, IF dissociation, and fMet-tRNAfMet positioning during 70S EC formation. Our results demonstrate the power of TR cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

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.

Modeling Transcription Initiation By Bacterial RNA Polymerase

Transcription initiation is a highly regulated step of gene expression. Here, we discuss the series of large conformational changes set in motion by initial specific binding of bacterial RNA polymerase (RNAP) to promoter DNA and their relevance for regulation. Bending and wrapping of the upstream duplex facilitates bending of the downstream duplex into the active site cleft, nucleating opening of 13 bp in the cleft. The rate-determining opening step, driven by binding free energy, forms an unstable open complex, probably with the template strand in the active site. At some promoters, this initial open complex is greatly stabilized by rearrangements of the discriminator region between the −10 element and +1 base of the nontemplate strand and of mobile in-cleft and downstream elements of RNAP. The rate of open complex formation is regulated by effects on the rapidly-reversible steps preceding DNA opening, while open complex lifetime is regulated by effects on the stabilization of the initial open complex. Intrinsic DNA opening-closing appears less regulated. This noncovalent mechanism and its regulation exhibit many analogies to mechanisms of enzyme catalysis.

When the ribosome encounters a stop codon, it recruits a release factor (RF) to hydrolyze the ester bond between the peptide chain and tRNA. This chapter discusses the role of the compact structures of RF1 and RF2 for fast and accurate stop-codon recognition in translation termination. It utilizes time-resolved cryogenic electron microscopy (cryo-EM) to visualize transient compact forms of RF1 and RF2 at 3.5 and 4 Å resolution, respectively, in the codon-recognizing ribosome complex on the native pathway. Cryo-EM showed that ribosome-bound RFs have extended structures, suggesting that RFs are compact when entering the ribosome and then extend their structures upon stop codon recognition. Most intracellular functions are carried out by proteins, assembled as chains of peptide-bond linked amino acid residues on large ribonucleoprotein particles called ribosomes. The chapter suggest that the selective advantage of the compact RF-form is that it allows for rapid factor binding into and dissociation from an accuracy maximizing pre-accommodation state.

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

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

Binding of activators to upstream DNA sequences regulates transcription initiation by affecting the stability of the initial RNA polymerase (RNAP)-promoter complex and/or the rate of subsequent conformational changes required to form the open complex (RP(O)). Here we observe that the presence of nonspecific upstream DNA profoundly affects an early step in formation of the transcription bubble. Kinetic studies with the lambdaP(R) promoter and Escherichia coli RNAP reveal that the presence of DNA upstream of base pair -47 greatly increases the rate of forming RP(O), without significantly affecting its rate of dissociation. We find that this increase is largely due to an acceleration of the rate-limiting step (isomerization) in RP(O) formation, a step that occurs after polymerase binds. Footprinting experiments reveal striking structural differences downstream of the transcription start site (+1) in the first kinetically significant intermediate when upstream DNA is present. On the template strand, the DNase I downstream boundary of this early intermediate is +20 when upstream DNA is present but is shortened by approximately two helical turns when upstream DNA beyond -47 is removed. KMnO(4) footprinting reveals an identical initiation bubble (-11 to +2), but unusual reactivity of template strand upstream cytosines (-12, -14, and -15) on the truncated promoter. Based on this work, we propose that early wrapping interactions between upstream DNA and the polymerase exterior strongly affect the events that control entry and subsequent unwinding of the DNA start site in the jaws of polymerase.

Transcription by all RNA polymerases (RNAPs) requires a series of large-scale conformational changes to form the transcriptionally competent open complex RP(o). At the lambdaP(R) promoter, Escherichia coli sigma(70) RNAP first forms a wrapped, closed 100 bp complex I(1). The subsequent step opens the entire DNA bubble, creating the relatively unstable (open) complex I(2). Additional conformational changes convert I(2) to the stable RP(o). Here we probe these events by dissecting the effects of Na(+) salts of Glu(-), F(-), and Cl(-) on each step in this critical process. Rapid mixing and nitrocellulose filter binding reveal that the binding constant for I(1) at 25 degrees C is approximately 30-fold larger in Glu(-) than in Cl(-) at the same Na(+) concentration, with the same log-log salt concentration dependence for both anions. In contrast, both the rate constant and equilibrium constant for DNA opening (I(1) to I(2)) are only weakly dependent on salt concentration, and the opening rate constant is insensitive to replacement of Cl(-) with Glu(-). These very small effects of salt concentration on a process (DNA opening) that is strongly dependent on salt concentration in solution may indicate that the backbones of both DNA strands interact with polymerase throughout the process and/or that compensation is present between ion uptake and release. Replacement of Cl(-) with Glu(-) or F(-) at 25 degrees C greatly increases the lifetime of RP(o) and greatly reduces its salt concentration dependence. By analogy to Hofmeister salt effects on protein folding, we propose that the excluded anions Glu(-) and F(-) drive the folding and assembly of the RNAP clamp/jaw domains in the conversion of I(2) to RP(o), while Cl(-) does not. Because the Hofmeister effect of Glu(-) or F(-) largely compensates for the destabilizing Coulombic effect of any salt on the binding of this assembly to downstream promoter DNA, RP(o) remains long-lived even at 0.5 M Na(+) in Glu(-) or F(-) salts. The observation that Esigma(70) RP(o) complexes are exceedingly long-lived at moderate to high Glu(-) concentrations argues that Esigma(70) RNAP does not dissociate from strong promoters in vivo when the cytoplasmic glutamate concentration increases during osmotic stress.

Transcription elongation requires the continuous incorporation of ribonucleotide triphosphates into a growing transcript. RNA polymerases (RNAPs) are able to processively synthesize a growing RNA chain via translocation of the RNAP enzyme along its nucleic acid template strand after each nucleotide addition cycle. In this work, a time-resolved Raman spectroscopic analysis of nucleotide addition in single crystals of the Thermus thermophilus elongation complex (TthEC) is reported. When [(13)C,(15)N]GTP (*GTP) is soaked into crystals of the TthEC, large reversible changes in the Raman spectrum that are assigned to protein and nucleic acid conformational events during a single-nucleotide incorporation are observed. The *GTP population in the TthEC crystal reaches a stable population at 37 min, while substantial and reversible protein conformational changes (mainly ascribed to changes in α-helical Raman features) maximize at approximately 50 min. At the same time, changes in nucleic acid bases and phosphodiester backbone Raman marker bands occur. Catalysis begins at approximately 65-70 min, soon after the maximal protein and DNA changes, and is monitored via the decline in a triphosphate vibrational Raman mode from *GTP. The Raman data indicate that approximately 40% of the total triphosphate population, present as *GTP, reacts in the crystal. This may suggest that a second population of noncovalently bound *GTP resides in a site distinct from the catalytic site. The data reported here are an extension of our recent work on the elongation complex (EC) of a bacterial RNAP, Thermus thermophilus (Tth), where Raman spectroscopy and polyacrylamide gel electrophoresis were employed to monitor incorporation and misincorporation in single TthEC crystals [Antonopoulos, I. H., et al. (2015) Biochemistry 54, 652-665]. Therefore, the initial study establishes the groundwork for this study. In contrast to our previous study, in which incorporation takes place very rapidly inside the crystals, the data on this single crystal exhibit a slower time regime, which allows the dissection of the structural dynamics associated with GMP incorporation within the TthEC crystal.

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.

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.

Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin

To maintain metabolic homeostasis, enzymes must adapt to fluctuating nutrient levels through mechanisms beyond gene expression. Here, we demonstrate that human glutamine synthetase (GS) can reversibly polymerize into filaments aided by a composite binding site formed at the filament interface by the product, glutamine. Time-resolved cryo-electron microscopy (cryo-EM) confirms that glutamine binding stabilizes these filaments, which in turn exhibit reduced catalytic specificity for ammonia at physiological concentrations. This inhibition appears induced by a conformational change that remodulates the active site loop ensemble gating substrate entry. Metadynamics ensemble refinement revealed >10 Å conformational range for the active site loop and that the loop is stabilized by transient contacts. This disorder is significant, as we show that the transient contacts which stabilize this loop in a closed conformation are essential for catalysis both in vitro and in cells. We propose that GS filament formation constitutes a negative-feedback mechanism, directly linking product concentration to the structural and functional remodeling of the enzyme.

Transcriptional fidelity, which prevents the misincorporation of incorrect nucleoside monophosphates in RNA, is essential for life. Results from molecular dynamics (MD) simulations of eukaryotic RNA polymerase (RNAP) II and bacterial RNAP with experimental data suggest that fidelity may involve as many as five checkpoints. Using MD simulations, the effects of different active site NTPs in both open and closed trigger loop (TL) structures of RNAPs are compared. Unfavorable initial binding of mismatched substrates in the active site with an open TL is proposed to be the first fidelity checkpoint. The leaving of an incorrect substrate is much easier than a correct one energetically from the umbrella sampling simulations. Then, the closing motion of the TL, required for catalysis, is hindered by the presence of mismatched NTPs. Mismatched NTPs also lead to conformational changes in the active site, which perturb the coordination of magnesium ions and likely affect the ability to proceed with catalysis. This step appears to be the most important checkpoint for deoxy-NTP discrimination. Finally, structural perturbations in the template DNA and the nascent RNA in the presence of mismatches likely hinder nucleotide addition and provide the structural foundation for backtracking followed by removing erroneously incorporated nucleotides during proofreading.