RNA Polymerase Pausing during Initial Transcription

In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause (‘‘initiation pause,’’ �20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA

Selection against Spurious Promoter Motifs Correlates with Translational Efficiency across Bacteria

Because binding of RNAP to misplaced sites could compromise the efficiency of transcription, natural selection for the optimization of gene expression should regulate the distribution of DNA motifs capable of RNAP-binding across the genome. Here we analyze the distribution of the −10 promoter motifs that bind the σ70 subunit of RNAP in 42 bacterial genomes. We show that selection on these motifs operates across the genome, maintaining an over-representation of −10 motifs in regulatory sequences while eliminating them from the nonfunctional and, in most cases, from the protein coding regions. In some genomes, however, −10 sites are over-represented in the coding sequences; these sites could induce pauses effecting regulatory roles throughout the length of a transcriptional unit. For nonfunctional sequences, the extent of motif under-representation varies across genomes in a manner that broadly correlates with the number of tRNA genes, a good indicator of translational speed and growth rate. This suggests that minimizing the time invested in gene transcription is an important selective pressure against spurious binding. However, selection against spurious binding is detectable in the reduced genomes of host-restricted bacteria that grow at slow rates, indicating that components of efficiency other than speed may also be important. Minimizing the number of RNAP molecules per cell required for transcription, and the corresponding energetic expense, may be most relevant in slow growers. These results indicate that genome-level properties affecting the efficiency of transcription and translation can respond in an integrated manner to optimize gene expression. The detection of selection against promoter motifs in nonfunctional regions also confirms previous results indicating that no sequence may evolve free of selective constraints, at least in the relatively small and unstructured genomes of bacteria

Distribution of high mobility group proteins 1/2, E and 14/17 and linker histones H1 and H5 on transcribed and non-transcribed regions of chicken erythrocyte chromatin.

Quantitative analysis of distribution of chromosomal proteins on single copy DNA sequences has been further developed. Our approach consists of DNA-protein crosslinking within whole cells or isolated nuclei, specific immunoaffinity isolation of crosslinked complexes via protein and identification of crosslinked DNA by hybridisation with single-stranded DNA probes. The present study shows that transcribed chromatin of chicken embryonic erythrocyte beta globin gene is characterized by about 1.5-2.5-fold higher density of HMG 14/17 and 2-fold lower density of H1 and H5 as compared with non-transcribed chromatin of ovalbumin and lysozyme genes, whereas HMG 1/2, E proteins were equally distributed between DNA of both transcribed and non-transcribed genes. The depletion of H1/H5 in beta globin sequences was verified by the 'protein image' hybridisation technique (1). The DNase I hypersensitive site located 5' upstream from beta globin gene is deficient in all the proteins assayed, what implies a drastic disruption in the nucleosomal array. Minor quantitative changes of protein pattern suggest transient local perturbation of the chromatin on transcription

RNA polymerase pausing during initial transcription

In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA

SOLEIL shining on the solution-state structure of biomacromolecules by synchrotron X-ray footprinting at the metrology beamline

Synchrotron X-ray footprinting complements the techniques commonly used to define the structure of molecules such as crystallography, small-angle X-ray scattering and nuclear magnetic resonance. It is remarkably useful in probing the structure and interactions of proteins with lipids, nucleic acids or with other proteins in solution, often better reflecting the in vivo state dynamics. To date, most X-ray footprinting studies have been carried out at the National Synchrotron Light Source, USA, and at the European Synchrotron Radiation Facility in Grenoble, France. This work presents X-ray footprinting of biomolecules performed for the first time at the X-ray Metrology beamline at the SOLEIL synchrotron radiation source. The installation at this beamline of a stopped-flow apparatus for sample delivery, an irradiation capillary and an automatic sample collector enabled the X-ray footprinting study of the structure of the soluble protein factor H (FH) from the human complement system as well as of the lipid-associated hydrophobic protein S3 oleosin from plant seed. Mass spectrometry analysis showed that the structural integrity of both proteins was not affected by the short exposition to the oxygen radicals produced during the irradiation. Irradiated molecules were subsequently analysed using high-resolution mass spectrometry to identify and locate oxidized amino acids. Moreover, the analyses of FH in its free state and in complex with complement C3b protein have allowed us to create a map of reactive solvent-exposed residues on the surface of FH and to observe the changes in oxidation of FH residues upon C3b binding. Studies of the solvent accessibility of the S3 oleosin show that X-ray footprinting offers also a unique approach to studying the structure of proteins embedded within membranes or lipid bodies. All the biomolecular applications reported herein demonstrate that the Metrology beamline at SOLEIL can be successfully used for synchrotron X-ray footprinting of biomolecules