Robert Landick - Publications
Genomic analysis of protein-DNA interactions in bacteria: insights into transcription and chromosome . H-NS and RNA polymerase: a love-hate relationship?. this fundamental process is the single RNA polymerase core enzyme (unlike Eukaryotes that have multiple . There is indeed an interesting relationship between H-NS and RNA polymerase as H-NS and RNA polymerase: A love- hate. causes titration of RNA polymerase and a global downshift in host gene expression. H-NS and RNA polymerase: a love-hate relationship?.
The bridge helix coordinates movements of modules in RNA polymerase. RNA polymerase mutants found through adaptive evolution reprogram Escherichia coli for optimal growth in minimal media. Complete structural model of Escherichia coli RNA polymerase from a hybrid approach. Roles for the transcription elongation factor NusA in both DNA repair and damage tolerance pathways in Escherichia coli.
Taking phage integration to the next level as a genetic tool for mycobacteria. Role of the RNA polymerase trigger loop in catalysis and pausing. Rho directs widespread termination of intragenic and stable RNA transcription. Two structurally independent domains of E. Transcriptional pausing without backtracking. Functional divergence in the growing family of RNA polymerases. Regulator trafficking on bacterial transcription units in vivo. Functional specialization of transcription elongation factors.
Organization and evolution of the biological response to singlet oxygen stress. Applied force reveals mechanistic and energetic details of transcription termination. Rapid isolation and identification of bacteriophage T4-encoded modifications of Escherichia coli RNA polymerase: Journal of Proteome Research.
Integrating systems and synthetic biology to optimize biofuel pathways in microorganisms Aiche Annual Meeting, Conference Proceedings. A central role of the RNA polymerase trigger loop in active-site rearrangement during transcriptional pausing.
Structural basis for substrate loading in bacterial RNA polymerase.
H-NS and RNA polymerase: a love-hate relationship?
Direct versus limited-step reconstitution reveals key features of an RNA hairpin-stabilized paused transcription complex. Real-time footprinting of DNA in the first kinetically significant intermediate in open complex formation by Escherichia coli RNA polymerase. The regulatory roles and mechanism of transcriptional pausing Biochemical Society Transactions.
The role of the lid element in transcription by E. Pulling on the nascent RNA during transcription does not alter kinetics of elongation or ubiquitous pausing. Sequence-resolved detection of pausing by single RNA polymerase molecules. Functional interplay between the jaw domain of bacterial RNA polymerase and allele-specific residues in the product RNA-binding pocket. Sigma and RNA polymerase: Direct observation of base-pair stepping by RNA polymerase. Inhibition of RNA polymerase by streptolydigin: Immobilization of Escherichia coli RNA polymerase and location of binding sites by use of chromatin immunoprecipitation and microarrays.
Landick RC, et al. An open letter to Elias Zerhouni. A hydrophobic patch on the flap-tip helix of E. Active-site dynamics in RNA polymerases. Diversity in the rates of transcript elongation by single RNA polymerase molecules. M 1 Toulokhonov I, Landick R. The flap domain is required for pause RNA hairpin inhibition of catalysis by RNA polymerase and can modulate intrinsic termination. Backtracking by single RNA polymerase molecules observed at near-base-pair resolution.
Tethering sigma70 to RNA polymerase reveals high in vivo activity of sigma factors and sigmadependent pausing at promoter-distal locations. Ubiquitous transcriptional pausing is independent of RNA polymerase backtracking.
PDF of The love-hate relationship between bacterial polysaccharides and the host immune system
A new class of bacterial RNA polymerase inhibitor affects nucleotide addition. Structure of microcin J25, a peptide inhibitor of bacterial RNA polymerase, is a lassoed tail. Journal of the American Chemical Society. RNA polymerase mutations that impair conversion to a termination-resistant complex by Q antiterminator proteins. Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions.
Mutations of bacterial RNA polymerase leading to resistance to microcin j M 1 Artsimovitch ILandick R. Binding of the initiation factor sigma 70 to core RNA polymerase is a multistep process. RNA polymerase clamps down Cell. RNA polymerases from Bacillus subtilis and Escherichia coli differ in recognition of regulatory signals in vitro Journal of Bacteriology. Protein Expression and Purification. Single-molecule study of transcriptional pausing and arrest by E.
Direct localization of a beta-subunit domain on the three-dimensional structure of Escherichia coli RNA polymerase. Nonequilibrium mechanism of transcription termination from observations of single RNA polymerase molecules. Folding of a large ribozyme during transcription and the effect of the elongation factor NusA. Shifting RNA polymerase into overdrive Science. Force and velocity measured for single molecules of RNA polymerase. Interaction of a nascent RNA structure with RNA polymerase is required for hairpin-dependent transcriptional pausing but not for transcript release Genes and Development.
Information processing by RNA polymerase: RNA polymerase as a molecular motor. Tethering of the large subunits of Escherichia coli RNA polymerase.
Multiple interactions stabilize a single paused transcription intermediate in which hairpin to 3' end spacing distinguishes pause and termination pathways Journal of Molecular Biology.
Stretching DNA with optical tweezers.
The love-hate relationship between bacterial polysaccharides and the host immune system
RNA polymerase slides home: Pause and termination site recognition Cell. Nuclease cleavage of the upstream half of the nontemplate strand DNA in an Escherichia coli transcription elongation complex causes upstream translocation and transcriptional arrest Journal of Biological Chemistry.
Quantitative analysis of transcriptional pausing by Escherichia coli RNA polymerase: Isolation, purification, and in vitro characterization of recessive- lethal-mutant RNA polymerases from Escherichia coli Journal of Bacteriology. The shrewd grasp of RNA polymerase. Amino acid substitutions in the two largest subunits of Escherichia coli RNA polymerase that suppress a defective Rho termination factor affect different parts of the transcription complex.
Single-molecule kinetic studies on DNA transcription and transcriptional regulation.
Streptolydigin-resistant mutants in an evolutionarily conserved region of the beta' subunit of Escherichia coli RNA polymerase. Transcription against an applied force. GreA-induced transcript cleavage is accompanied by reverse translocation to a different transcription complex conformation. GreA-induced transcript cleavage in transcription complexes containing Escherichia coli RNA polymerase is controlled by multiple factors, including nascent transcript location and structure.
Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule. Thus, LeuO is also a negative autoregulator Stratmann et al. BglJ-RcsB is a heterodimer that activates transcription of various loci in E. BglJ-RcsB consists of RcsB, the response regulator of the Rcs two-component phosphorelay system Majdalani and Gottesman,and BglJ, which has initially been found as an activator of the bgl operon Giel et al.
A Regulation of leuO by interlocked double-positive and negative feedback loops.
Mutual positive regulation represents a double-positive feedback loop. To monitor leuO transcription a PleuO mVenus fusion was constructed by replacement of the native leuO gene with mVenus. Expression of bglJ and leuO, respectively, was induced with gradually increasing concentrations of the inducers arabinose and IPTG, respectively. This double-positive feedback loop is interlocked with a negative feedback loop which is based on negative autoregulation by LeuO Figure 1.
Such a network motif can function like a switch that is stable both in the OFF as well as in the ON state. Often an external signal locks such feedback loops in one state. Further, bi-stability resulting in population heterogeneity and oscillation can be based on interlocked positive and negative feedback loops Angeli et al. In this study we addressed the antagonistic regulation of leuO transcription by BglJ-RcsB and LeuO, which is presumably a crucial element in the complex control of leuO expression.
For quantitative and single-cell expression analysis, we established a reporter fusion of the leuO promoter region PleuO to mVenus and expressed bglJ and leuO in trans using tightly controlled and gradually inducible plasmidic expression systems.
Expression analyses of the PleuO mVenus reporter at steady state growth conditions revealed uniform expression. The data are in agreement with a straightforward model of antagonistic regulation by the two regulators that act independently of each other.
To address regulation of leuO transcription that is directed by at least two promoters PleuO in dependence of the concentrations of BglJ and LeuO, a suitable experimental system was established. Second, BglJ and LeuO were ectopically expressed from two different sets of plasmids.
The genes encoding the AraC and the LacI regulators, respectively, are also carried on these plasmids. Likewise, the arabinose regulon was modified to ensure a gradual induction of the PBAD promoter with arabinose, as described before Khlebnikov et al. Briefly, the PBAD promoter is known to have a stochastic behavior when induced with arabinose.
This stochastic behavior is caused by the araE and araFGH genes encoding the arabinose transporters, because induction of the transporter genes by arabinose leads to a higher arabinose uptake and thus positive feedback Siegele and Hu, ; Megerle et al.
In addition, a negative feedback caused by fermentation of intracellular arabinose through the AraBAD enzymes leads to a non-gradual induction Siegele and Hu, Further, the low affinity arabinose transporter araE was put under the control of constitutive promoter Pcp8, as described Khlebnikov et al.
Using this strain the expression level of PleuO mVenus was measured by flow-cytometry to quantify the cellular fluorescence in the population. Further, to ensure steady state conditions, cultures were grown in nutrient-poor tryptone medium. In this medium cultures that were inoculated from fresh overnight cultures to OD of 0.
Expression of bglJ was either not induced or induced by gradually increasing inducer concentrations. Activation of leuO transcription by BglJ. Expression was analyzed after 5 h of growth in tryptone medium without and with indicated inducer concentrations at an optical density OD of approximately 0.
PDB 1ni8 citation summary ‹ Protein Data Bank in Europe (PDBe) ‹ EMBL-EBI
Yellow fluorescence X-axis is given in arbitrary units and the Y-axis gives the number of cells that were counted. The median of the fluorescence intensity is given in the upper right corner of the graph. B Plot of the median fluorescence values that are shown in C solid line with filled dots and D solid line with filled squares PleuO mVenus and dashed line with open squares PleuO leuO:: The arabinose concentration used for induction of bglJ expression is given underneath the panels.
Shown are representative data. Flow cytometry revealed a slight increase in PleuO mVenus expression at low levels of induction of plasmidic leuO Figure 3. The data seem in agreement with weak positive autoregulation that was reported previously Fang and Wu, ; Chen et al.
Autoregulation of leuO transcription. Fluorescence expression levels directed by the PleuO mVenus fusion were determined by flow cytometry. The fluorescence median is plotted against the inducer concentration. Expression was analyzed by flow cytometry after 5 h of growth in trypton medium, IPTG, and arabinose were added at the indicated concentrations.
To this end, the PleuO mVenus reporter strain U69 was transformed with the two sets of leuO and bglJ expressing plasmids. Since the PBAD bglJ plasmid does not allow gradual activation, this plasmid set does not seem suitable for gradual induction of both regulators.
A The median fluorescence is plotted against the arabinose concentration used for induction of bglJ. Each line graph represents the set of data obtained of cultures grown with the specified IPTG concentration used for induction of leuO. B Flow cytometry data of cultures grown with increasing arabinose rows and IPTG columns concentration. Plotted in each panel are the cell counts against the fluorescence intensity.
The fluorescence distribution in each panel is in agreement with uniform expression within the population. The fluorescence median that is plotted in A is given within each panel.