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The ribonuclease FttA (also known as aCPSF and aCPSF1) mediates factor-dependent transcription termination in archaea1–3. Here we report the structure of a Thermococcus kodakarensis transcription pre-termination complex comprising FttA, Spt4, Spt5 and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic centre and that enables endonucleolytic cleavage of RNA by FttA, followed by 5′→3′ exonucleolytic cleavage of RNA by FttA and concomitant 5′→3′ translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination. The structure further reveals that Spt5 bridges FttA and the TEC, explaining how Spt5 stimulates FttA-dependent termination. The results reveal functional analogy between bacterial and archaeal factor-dependent termination, functional homology between archaeal and eukaryotic factor-dependent termination, and fundamental mechanistic similarities in factor-dependent termination in bacteria, archaea, and eukaryotes.Cryo-electron microscopy structures of the Thermococcus kodakarensis transcription pre-termination complex suggest a mechanism by which the archaeal termination factor FttA applies mechanical force to a transcription elongation complex to trigger termination, and reveal similarities in factor-dependent termination in bacteria, archaea, and eukaryotes. [ABSTRACT FROM AUTHOR]

Rho is a ring-shaped hexameric ATP-dependent molecular motor. Together with the transcription elongation factor NusG, Rho mediates factor-dependent transcription termination and transcription–translation-coupling quality control in Escherichia coli1–4. Here we report the preparation of complexes that are functional in factor-dependent transcription termination from Rho, NusG, RNA polymerase (RNAP), and synthetic nucleic acid scaffolds, and we report cryogenic electron microscopy structures of the complexes. The structures show that functional factor-dependent pre-termination complexes contain a closed-ring Rho hexamer; have RNA threaded through the central channel of Rho; have 60 nucleotides of RNA interacting sequence-specifically with the exterior of Rho and 6 nucleotides of RNA interacting sequence-specifically with the central channel of Rho; have Rho oriented relative to RNAP such that ATP-dependent translocation by Rho exerts mechanical force on RNAP; and have NusG bridging Rho and RNAP. The results explain five decades of research on Rho and provide a foundation for understanding Rho’s function.Structures presented in this study confirm decades of genetic and biochemical evidence for the mechanism of Rho-dependent termination in bacteria. [ABSTRACT FROM AUTHOR]

We report a metabolomic analysis of Streptomyces sp. ID38640, a soil isolate that produces the bacterial RNA polymerase inhibitor pseudouridimycin. The analysis was performed on the wild type, on three newly constructed and seven previously reported mutant strains disabled in different genes required for pseudouridimycin biosynthesis. The results indicate that Streptomyces sp. ID38640 is able to produce, in addition to lydicamycins and deferroxiamines, as previously reported, also the lassopeptide ulleungdin, the non-ribosomal peptide antipain and the osmoprotectant ectoine. The corresponding biosynthetic gene clusters were readily identified in the strain genome. We also detected the known compound pyridindolol, for which we propose a previously unreported biosynthetic gene cluster, as well as three families of unknown metabolites. Remarkably, the levels of most metabolites varied strongly in the different mutant strains, an observation that enabled detection of metabolites unnoticed in the wild type. Systematic investigation of the accumulated metabolites in the ten different pum mutants identified shed further light on pseudouridimycin biosynthesis. We also show that several Streptomyces strains, able to produce pseudouridimycin, have distinct genetic relationship and metabolic profile with ID38640. [ABSTRACT FROM AUTHOR]

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 α-subunit carboxy-terminal domain and on the presence and length of the RNAP α-subunit interdomain linker. Our results indicate that the extensive stable DNA wrapping observed previously in the RNAP–promoter open complex at the λ PR promoter is not a general feature of RNAP–promoter open complexes. [ABSTRACT FROM AUTHOR]

Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of a CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching protein motions. As a result, the second cAMP binding incurs a pronounced conformational entropic penalty that is entirely responsible for the observed cooperativity. The results provide strong support for the existence of purely dynamics-driven allostery. [ABSTRACT FROM AUTHOR]

Reports that single-molecule nanomanipulation of supercoiled DNA permits measurement of spatial and temporal parameters of protein-DNA interactions that affect DNA topology. Methods involved in the process; Reagent materials needed in the process; Preparation procedure for the preparation of DNA constructs.

The s70 subunit of RNA polymerase plays an essential role in transcription initiation. In addition, s70 has a critical regulatory role during transcription elongation at the bacteriophage ? late promoter, ?PR'. At this promoter, s70 mediates a pause in early elongation through contact with a DNA sequence element in the initially transcribed region that resembles a promoter -10 element. Here we provide evidence that s70 also mediates a pause in early elongation at the lac promoter (plac). Like that at ?PR', the pause at plac is facilitated by a sequence element in the initially transcribed region that resembles a promoter -10 element. Using biophysical analysis, we demonstrate that the pause-inducing sequence element at plac stabilizes the interaction between s70 and the remainder of the transcription elongation complex. Bioinformatic analysis suggests that promoter-proximal s70-dependent pauses may play a role in the regulation of many bacterial promoters. [ABSTRACT FROM AUTHOR]

Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase. At Class II CRPdependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase α subunit C-terminal domain (αCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase a subunit N-terminal domain that facilitates subsequent promoter opening. We have used random mutagenesis and alanine scanning to identify determinants within αCTD for transcription activation at a Class II CRP-dependent promoter. Our results indicate that Class II CRP-dependent transcription requires the side chains of residues 265, 271, 285–288 and 317. Residues 285–288 and 317 comprise a discrete 20310 Å surface on αCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between a subunits and CRP, but do not affect DNA binding by a subunits. We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in &lpha;CTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for αCTD-CRP interaction in Class I CRP-dependent transcription. [ABSTRACT FROM AUTHOR]

Extracytoplasmic (ECF) σ factors, the largest class of alternative σ factors, are related to primary σ factors, but have simpler structures, comprising only two of six conserved functional modules in primary σ factors: region 2 (σR2) and region 4 (σR4). Here, we report crystal structures of transcription initiation complexes containing Mycobacterium tuberculosis RNA polymerase (RNAP), M. tuberculosis ECF σ factor σL, and promoter DNA. The structures show that σR2 and σR4 of the ECF σ factor occupy the same sites on RNAP as in primary σ factors, show that the connector between σR2 and σR4 of the ECF σ factor-although shorter and unrelated in sequence-follows the same path through RNAP as in primary σ factors, and show that the ECF σ factor uses the same strategy to bind and unwind promoter DNA as primary σ factors. The results define protein-protein and protein-DNA interactions involved in ECF-σ-factor-dependent transcription initiation. No structural data have been available for RNA polymerase holoenzymes or transcription initiation complexes that contain extracytoplasmic σ factors. Here the authors report the crystal structures of transcription initiation complexes comprising Mycobacterium tuberculosis RNA polymerase, extracytoplasmic σ factor σL and promoter DNA. [ABSTRACT FROM AUTHOR]

Transcription by RNA polymerase (RNAP) is interspersed with sequence-dependent pausing. The processes through which paused states are accessed and stabilized occur at spatiotemporal scales beyond the resolution of previous methods, and are poorly understood. Here, we combine high-resolution optical trapping with improved data analysis methods to investigate the formation of paused states at enhanced temporal resolution. We find that pause sites reduce the forward transcription rate of nearly all RNAP molecules, rather than just affecting the subset of molecules that enter long-lived pauses. We propose that the reduced rates at pause sites allow time for the elongation complex to undergo conformational changes required to enter long-lived pauses. We also find that backtracking occurs stepwise, with states backtracked by at most one base pair forming quickly, and further backtracking occurring slowly. Finally, we find that nascent RNA structures act as modulators that either enhance or attenuate pausing, depending on the sequence context. [ABSTRACT FROM AUTHOR]