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WT 110901_SN865_B_L006_R1_GQC-28- ATCACG.fastq.gz 100 19.624.852 1,29

llumina fastq format 4 lines for each sequence: 1- Unique identifier, with the following format: @::::#/ 2- Sequence (A, T, C ,G or N (undetermined) only) 3- Orientation (always forward without mapping) 4- Quality value for each base, corresponding to a Phred-like score encoded in ASCII format, with an offset of of 33 (e.g. “J” gives a value of 41) and is in accordance with sanger FASTQ format. The sequence file is compressed as tar.gz archive. The archive can be uncompressed using a tar -zxvf command.

SEEK ID: https://fairdomhub.org/data_files/914?version=1

Filename: 110901_SN865_B_L006_R1_GQC-28-ATCACG_WT-D39.fastq.gz

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Jan-Willem Veening

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Jan-Willem Veening

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Created: 23rd Nov 2011 at 16:37

Last updated: 26th Aug 2014 at 12:20

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Version 1 (earliest) Created 23rd Nov 2011 at 16:37 by Jan-Willem Veening

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Publications (21)

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Jan-Willem Veening

Projects: Noisy-Strep

Institutions: University of Groningen

Expertise: Microbiology, Genetics, Molecular Biology, Single Cell analysis, Bacillus subtilis, Bacterial Cell Biology, Molecular microbiology, Medical microbiology, Streptococcus pneumoniae

Tools: Microbiology, Biochemistry, Genetics, Genetic modification, Transcriptomics, Fluorecence based reporter gene analyses/single cell analyses, Molecular biology techniques (RNA/DNA/Protein)

The Veening lab is interested in phenotypic bi-stability in Streptococcus pneumoniae and its importance in virulence of this human pathogen.

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SysMO

SysMO is a European transnational funding and research initiative on "Systems Biology of Microorganisms".

The goal pursued by SysMO was to record and describe the dynamic molecular processes going on in unicellular microorganisms in a comprehensive way and to present these processes in the form of computerized mathematical models.

Systems biology will raise biomedical and biotechnological research to a new quality level and contribute markedly to progress in understanding. Pooling European research ...

Projects: BaCell-SysMO, COSMIC, SUMO, KOSMOBAC, SysMO-LAB, PSYSMO, SCaRAB, MOSES, TRANSLUCENT, STREAM, SulfoSys, SysMO DB, SysMO Funders, SilicoTryp, Noisy-Strep

Web page: http://sysmo.net/

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Noisy-Strep

Bistable switches are the key elements of the regulatory networks governing cell development, differentiation and life-strategy decisions. Transcriptional noise is a main determinant that causes switching between different states in bistable systems. By using the human pathogen Streptococcus pneumoniae as a model bacterium, we will investigate how transcriptional fidelity and processivity influence (noisy) gene expression and participate in bistability. To study this question, we will use both ...

Programme: SysMO

Public web page: http://www.sysmo.net/index.php?index=163

Organisms: Streptococcus pneumoniae

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Wetlab approach to transcription fidelity

Noisy-Strep

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Jan-Willem Veening

Handling and manipulation of S. pneumoniae using molecular, cell biological and genetic tools.

Submitter: Jan-Willem Veening

Studies: Automated time-lapse microscopy, Chromosome segregation in S. pneumoniae, Investigation of bacterial transcription fidelity and processivity, The role of transcription factor GreA in transcription fidelity and proc...

Assays: Kinetics of misincorporation and proofreading by bacterial RNA polymerase, Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae usin..., ParB-GFP ChIP on chip, RNA-Seq

Snapshots: No snapshots

Created: 8th Feb 2011 at 11:40, Last updated: 22nd Nov 2011 at 19:12

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The role of transcription factor GreA in transcription fidelity and processivity in Streptococcus pneumoniae

Noisy-Strep

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Jan-Willem Veening

For cells to accurately read out the genomic content, high fidelity during transcription is required. This is mainly established by the accuracy of the active centre of RNA polymerase (RNAP). Based on in vitro experiments with Escherichia coli RNAP it was also suggested that proofreading of transcription via RNA hydrolysis by RNAP may contribute to overall fidelity and processivity. RNAP’s intrinsic cleavage activity is stimulated by the highly conserved Gre factors suggesting that Gre factors ...

Submitter: Jan-Willem Veening

Investigation: Wetlab approach to transcription fidelity

Assays: RNA-Seq

Snapshots: No snapshots

Created: 23rd Nov 2011 at 15:39, Last updated: 26th Aug 2014 at 12:20

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RNA-Seq

Noisy-Strep

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Jan-Willem Veening

S.pneumoniae D39 cells (wild type and delta greA) were grown in C+Y medium and cells were harvested for total RNA isolation at mid-exponential growth (OD600nm 0.3 for wt, 0.25 for delta greA). Total RNA was isolated as described before (Kloosterman et al 2006). The total RNA samples were examined by capillary electrophoresis. dephosphorylated with antarctic phosphatase followed by treatment with polynucleotide kinase (PNK). Afterwards, samples were poly(A)-tailed using poly(A) polymerase. Then a ...

Submitter: Jan-Willem Veening

Assay type: Transcriptomics

Technology type: Technology Type

Investigation: Wetlab approach to transcription fidelity

Study: The role of transcription factor GreA in transc...

Organisms: No organisms

SOPs: No SOPs

Data files: RNA-seq delta greA, RNA-seq wild type

Snapshots: No snapshots

Created: 23rd Nov 2011 at 15:57, Last updated: 8th Nov 2017 at 14:21

Showing 20 out of a possible 21 Publications Advanced Publications list for this Data file with search and filtering

Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae

Noisy-Strep

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Yulia Yuzenkova

Pamela Gamba

Sulman Shafeeq

Oscar Kuipers

Nikolay Zenkin

Jan-Willem Veening

Abstract (Expand)

Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such … pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in 'transcription traffic jams' on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes.

Authors: , , M. Herber, L. Attaiech, , , S. Klumpp, ,

Date Published: 6th Sep 2014

Publication Type: Not specified

PubMed ID: 25190458

Citation:

Created: 8th Sep 2014 at 13:31, Last updated: 8th Dec 2022 at 17:26

GraphAlignment: Bayesian pairwise alignment of biological networks

Noisy-Strep

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Johannes Berg

Abstract (Expand)

ABSTRACT: BACKGROUND: With increased experimental availability and accuracy of bio-molecular networks, tools for their comparative and evolutionary analysis are needed. A key component for such studies … is the alignment of networks. RESULTS: We introduce the Bioconductor package GraphAlignment for pairwise alignment of bio-molecular networks. The alignment incorporates information both from network vertices and network edges and is based on an explicit evolutionary model, allowing inference of all scoring parameters directly from empirical data. We compare the performance of our algorithm to an alternative algorithm, Graemlin 2.0.On simulated data, GraphAlignment outperforms Graemlin 2.0 in several benchmarks except for computational complexity. When there is little or no noise in the data, GraphAlignment is slower than Graemlin 2.0. It is faster than Graemlin 2.0 when processing noisy data containing spurious vertex associations. Its typical case complexity grows approximately as O(N^2.6). On empirical bacterial protein-protein interaction networks (PIN) and gene co-expression networks, GraphAlignment outperforms Graemlin 2.0 with respect to coverage and specificity, albeit by a small margin. On large eukaryotic PIN, Graemlin 2.0 outperforms GraphAlignment. CONCLUSIONS: The GraphAlignment algorithm is robust to spurious vertex associations, correctly resolves paralogs, and shows very good performance in identification of homologous vertices defined by high vertex and/or interaction similarity.

Authors: Michal Kolar, Jörn Meier, Ville Mustonen, Michael Lässig,

Date Published: 21st Nov 2012

Publication Type: Not specified

PubMed ID: 23171476

Citation:

Created: 18th Dec 2012 at 17:06, Last updated: 8th Dec 2022 at 17:26

Single cell analysis of gene expression patterns during carbon starvation in Bacillus subtilis reveals large phenotypic variation

Noisy-Strep

Abstract (Expand)

How cells dynamically respond to fluctuating environmental conditions depends on the architecture and noise of the underlying genetic circuits. Most work characterizing stress pathways in the model … bacterium Bacillus subtilis has been performed on bulk cultures using ensemble assays. However, investigating the single cell response to stress is important since noise might generate significant phenotypic heterogeneity. Here, we study the stress response to carbon source starvation and compare both population and single cell data. Using a top-down approach, we investigate the transcriptional dynamics of various stress-related genes of B. subtilis in response to carbon source starvation and to increased cell density. Our data reveal that most of the tested gene-regulatory networks respond highly heterogeneously to starvation and cells show a large degree of variation in gene expression. The level of highly dynamic diversification within B. subtilis populations under changing environments reflects the necessity to study cells at the single cell level.

Editor:

Date Published: 4th Oct 2012

Publication Type: Not specified

PubMed ID: 23033921

Citation:

Created: 18th Dec 2012 at 17:04, Last updated: 8th Dec 2022 at 17:26

Multiple active centers of multi-subunit RNA polymerases

Noisy-Strep

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Yulia Yuzenkova

Nikolay Zenkin

Abstract (Expand)

The active center of multi-subunit RNA polymerase consists of two modules, the Mg(2+) module, holding the catalytic Mg(2+) ion, and a module made of a flexible domain, the Trigger Loop. Uniquely, the … TL module can be substituted by alternative modules, thus changing the catalytic properties of the active center.

Authors: , Mohammad Roghanian,

Date Published: 10th Jul 2012

Publication Type: Not specified

PubMed ID: 22771945

Citation:

Created: 18th Dec 2012 at 17:02, Last updated: 8th Dec 2022 at 17:26

Hypothesis: emergence of translation as a result of RNA helicase evolution

Noisy-Strep

Abstract (Expand)

The origin of translation and the genetic code is one of the major mysteries of evolution. The advantage of templated protein synthesis could have been achieved only when the translation apparatus had … already become very complex. This means that the translation machinery, as we know it today, must have evolved towards some different essential function that subsequently sub-functionalised into templated protein synthesis. The hypothesis presented here proposes that translation originated as the result of evolution of a primordial RNA helicase, which has been essential for preventing dying out of the RNA organism in sterile double-stranded form. This hypothesis emerges because modern ribosome possesses RNA helicase activity that likely dates back to the RNA world. I hypothesise that codon-anticodon interactions of tRNAs with mRNA evolved as a mechanism used by RNA helicase, the predecessor of ribosomes, to melt RNA duplexes. In this scenario, peptide bond formation emerged to drive unidirectional movement of the helicase via a molecular ratchet mechanism powered by Brownian motion. I propose that protein synthesis appeared as a side product of helicase activity. The first templates for protein synthesis were functional RNAs (ribozymes) that were unwound by the helicase, and the first synthesised proteins were of random or non-sense sequence. I further suggest that genetic code emerged to avoid this randomness. The initial genetic code thus emerged as an assignment of amino acids to codons according to the sequences of the pre-existing RNAs to take advantage of the side products of RNA helicase function.

Editor:

Date Published: 28th Apr 2012

Publication Type: Not specified

PubMed ID: 22544085

Citation:

Created: 18th Dec 2012 at 17:01, Last updated: 8th Dec 2022 at 17:26

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP

Noisy-Strep

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Jan-Willem Veening

Abstract (Expand)

How the human pathogen Streptococcus pneumoniae coordinates cell-wall synthesis during growth and division to achieve its characteristic oval shape is poorly understood. The conserved eukaryotic-type … Ser/Thr kinase of S. pneumoniae, StkP, previously was reported to phosphorylate the cell-division protein DivIVA. Consistent with a role in cell division, GFP-StkP and its cognate phosphatase, GFP-PhpP, both localize to the division site. StkP localization depends on its penicillin-binding protein and Ser/Thr-associated domains that likely sense uncross-linked peptidoglycan, because StkP and PhpP delocalize in the presence of antibiotics that target the latest stages of cell-wall biosynthesis and in cells that have stopped dividing. Time-lapse microscopy shows that StkP displays an intermediate timing of recruitment to midcell: StkP arrives shortly after FtsA but before DivIVA. Furthermore, StkP remains at midcell longer than FtsA, until division is complete. Cells mutated for stkP are perturbed in cell-wall synthesis and display elongated morphologies with multiple, often unconstricted, FtsA and DivIVA rings. The data show that StkP plays an important role in regulating cell-wall synthesis and controls correct septum progression and closure. Overall, our results indicate that StkP signals information about the cell-wall status to key cell-division proteins and in this way acts as a regulator of cell division.

Authors: Katrin Beilharz, Linda Nováková, Daniela Fadda, Pavel Branny, Orietta Massidda,

Date Published: 21st Mar 2012

Publication Type: Not specified

PubMed ID: 22431591

Citation:

Created: 27th Mar 2012 at 13:43, Last updated: 8th Dec 2022 at 17:26

In vitro experimental system for analysis of transcription-translation coupling

Noisy-Strep

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Nikolay Zenkin

Abstract (Expand)

Transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. During transcription-translation, both machineries mutually affect each others' … functions, which is important for regulation of gene expression. Analysis of interactions between RNA polymerase (RNAP) and the ribosome, however, are limited due to the lack of an in vitro experimental system. Here, we report the development of an in vitro transcription coupled to translation system assembled from purified components. The system allows controlled stepwise transcription and simultaneous stepwise translation of the nascent RNA, and permits investigation of the interactions of RNAP with the ribosome, as well as the effects of translation on transcription and transcription on translation. As an example of usage of this experimental system, we uncover complex effects of transcription-translation coupling on pausing of transcription.

Authors: Daniel Castro-Roa,

Date Published: 3rd Jan 2012

Publication Type: Not specified

PubMed ID: 22210860

Citation:

Created: 5th Jan 2012 at 17:00, Last updated: 8th Dec 2022 at 17:26

Factor-independent transcription pausing caused by recognition of the RNA-DNA hybrid sequence

Noisy-Strep

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Yulia Yuzenkova

Nikolay Zenkin

Abstract (Expand)

Pausing of transcription is an important step of regulation of gene expression in bacteria and eukaryotes. Here we uncover a factor-independent mechanism of transcription pausing, which is determined … by the ability of the elongating RNA polymerase to recognize the sequence of the RNA-DNA hybrid. We show that, independently of thermodynamic stability of the elongation complex, RNA polymerase directly 'senses' the shape and/or identity of base pairs of the RNA-DNA hybrid. Recognition of the RNA-DNA hybrid sequence delays translocation by RNA polymerase, and thus slows down the nucleotide addition cycle through 'in pathway' mechanism. We show that this phenomenon is conserved among bacterial and eukaryotic RNA polymerases, and is involved in regulatory pauses, such as a pause regulating the production of virulence factors in some bacteria and a pause regulating transcription/replication of HIV-1. The results indicate that recognition of RNA-DNA hybrid sequence by multi-subunit RNA polymerases is involved in transcription regulation and may determine the overall rate of transcription elongation.

Authors: Aleksandra Bochkareva, , Vasisht R Tadigotla,

Date Published: 29th Nov 2011

Publication Type: Not specified

PubMed ID: 22124324

Citation:

Created: 5th Jan 2012 at 16:58, Last updated: 8th Dec 2022 at 17:26

Live Cell Imaging of <em>Bacillus subtilis</em> and <em>Streptococcus pneumoniae</em> using Automated Time-lapse Microscopy

Noisy-Strep

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Imke de Jong

Oscar Kuipers

Jan-Willem Veening

Abstract (Expand)

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of … single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see (1,2,3)). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy). Here, we provide a detailed description of a microscopy method used in several recent studies (4, 5, 6, 7), which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Authors: , Katrin Beilharz, ,

Date Published: 16th Aug 2011

Publication Type: Not specified

PubMed ID: 21841760

Citation:

Created: 21st Sep 2011 at 08:56, Last updated: 8th Dec 2022 at 17:26

A new basal promoter element recognized by RNA polymerase core enzyme

Noisy-Strep

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Yulia Yuzenkova

Nikolay Zenkin

Abstract (Expand)

Bacterial promoters are recognized by RNA polymerase (RNAP) σ subunit, which specifically interacts with the -10 and -35 promoter elements. Here, we provide evidence that the β' zipper, an … evolutionarily conserved loop of the largest subunit of RNAP core, interacts with promoter spacer, a DNA segment that separates the -10 and -35 promoter elements, and facilitates the formation of stable closed promoter complex. Depending on the spacer sequence, the proposed interaction of the β' zipper with the spacer can also facilitate open promoter complex formation and even substitute for interactions of the σ subunit with the -35 element. These results suggest that there exists a novel class of promoters that rely on interaction of the β' zipper with promoter spacer, along with or instead of interactions of σ subunit with the -35 element, for their activity. Finally, our data suggest that sequence-dependent interactions of the β' zipper with DNA can contribute to promoter-proximal σ-dependent RNAP pausing, a recently recognized important step of transcription control.

Authors: , Vasisht R Tadigotla, Konstantin Severinov,

Date Published: 26th Jul 2011

Publication Type: Not specified

PubMed ID: 21792175

Citation:

Created: 5th Jan 2012 at 16:56, Last updated: 8th Dec 2022 at 17:26

Nonlinear fitness landscape of a molecular pathway

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Johannes Berg

Abstract (Expand)

Genes are regulated because their expression involves a fitness cost to the organism. The production of proteins by transcription and translation is a well-known cost factor, but the enzymatic activity … of the proteins produced can also reduce fitness, depending on the internal state and the environment of the cell. Here, we map the fitness costs of a key metabolic network, the lactose utilization pathway in Escherichia coli. We measure the growth of several regulatory lac operon mutants in different environments inducing expression of the lac genes. We find a strikingly nonlinear fitness landscape, which depends on the production rate and on the activity rate of the lac proteins. A simple fitness model of the lac pathway, based on elementary biophysical processes, predicts the growth rate of all observed strains. The nonlinearity of fitness is explained by a feedback loop: production and activity of the lac proteins reduce growth, but growth also affects the density of these molecules. This nonlinearity has important consequences for molecular function and evolution. It generates a cliff in the fitness landscape, beyond which populations cannot maintain growth. In viable populations, there is an expression barrier of the lac genes, which cannot be exceeded in any stationary growth process. Furthermore, the nonlinearity determines how the fitness of operon mutants depends on the inducer environment. We argue that fitness nonlinearities, expression barriers, and gene-environment interactions are generic features of fitness landscapes for metabolic pathways, and we discuss their implications for the evolution of regulation.

Authors: Lilia Perfeito, Stéphane Ghozzi, , Karin Schnetz, Michael Lässig

Date Published: 21st Jul 2011

Publication Type: Not specified

PubMed ID: 21814515

Citation:

Created: 17th Jan 2012 at 11:36, Last updated: 8th Dec 2022 at 17:26

SMC is recruited to oriC by ParB and promotes chromosome segregation in Streptococcus pneumoniae

Noisy-Strep

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Jan-Willem Veening

Abstract (Expand)

Segregation of replicated chromosomes is an essential process in all organisms. How bacteria, such as the oval-shaped human pathogen Streptococcus pneumoniae, efficiently segregate their chromosomes … is poorly understood. Here we show that the pneumococcal homologue of the DNA-binding protein ParB recruits S. pneumoniae condensin (SMC) to centromere-like DNA sequences (parS) that are located near the origin of replication, in a similar fashion as was shown for the rod-shaped model bacterium Bacillus subtilis. In contrast to B. subtilis, smc is not essential in S. pneumoniae, and Δsmc cells do not show an increased sensitivity to gyrase inhibitors or high temperatures. However, deletion of smc and/or parB results in a mild chromosome segregation defect. Our results show that S. pneumoniae contains a functional chromosome segregation machine that promotes efficient chromosome segregation by recruitment of SMC via ParB. Intriguingly, the data indicate that other, as of yet unknown mechanisms, are at play to ensure proper chromosome segregation in this organism.

Authors: Anita Minnen, Laetitia Attaiech, Maria Thon, Stephan Gruber,

Date Published: 22nd Jun 2011

Publication Type: Not specified

PubMed ID: 21651626

Citation:

Created: 21st Sep 2011 at 08:55, Last updated: 8th Dec 2022 at 17:26

Controlled interplay between trigger loop and Gre factor in the RNA polymerase active centre

Noisy-Strep

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Yulia Yuzenkova

Nikolay Zenkin

Abstract (Expand)

The highly processive transcription by multi-subunit RNA polymerases (RNAP) can be interrupted by misincorporation or backtracking events that may stall transcription or lead to erroneous transcripts. … Backtracked/misincorporated complexes can be resolved via hydrolysis of the transcript. Here, we show that, in response to misincorporation and/or backtracking, the catalytic domain of RNAP active centre, the trigger loop (TL), is substituted by transcription factor Gre. This substitution turns off the intrinsic TL-dependent hydrolytic activity of RNAP active centre, and exchanges it to a far more efficient Gre-dependent mechanism of RNA hydrolysis. Replacement of the TL by Gre factor occurs only in backtracked/misincorporated complexes, and not in correctly elongating complexes. This controlled switching of RNAP activities allows the processivity of elongation to be unaffected by the hydrolytic activity of Gre, while ensuring efficient proofreading of transcription and resolution of backtracked complexes.

Authors: Mohammad Roghanian, ,

Date Published: 27th Jan 2011

Publication Type: Not specified

PubMed ID: 21266474

Citation:

Created: 10th Feb 2011 at 10:28, Last updated: 8th Dec 2022 at 17:26

DnaA and ORC: more than DNA replication initiators

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Jan-Willem Veening

Abstract (Expand)

Mutations in DNA replication initiator genes in both prokaryotes and eukaryotes lead to a pleiotropic array of phenotypes, including defects in chromosome segregation, cytokinesis, cell cycle regulation … and gene expression. For years, it was not clear whether these diverse effects were indirect consequences of perturbed DNA replication, or whether they indicated that DNA replication initiator proteins had roles beyond their activity in initiating DNA synthesis. Recent work from a range of organisms has demonstrated that DNA replication initiator proteins play direct roles in many cellular processes, often functioning to coordinate the initiation of DNA replication with essential cell-cycle activities. The aim of this review is to highlight these new findings, focusing on the pathways and mechanisms utilized by DNA replication initiator proteins to carry out a diverse array of cellular functions.

Authors: Graham Scholefield, , Heath Murray

Date Published: 27th Aug 2010

Publication Type: Not specified

PubMed ID: 21123069

Citation:

Created: 10th Feb 2011 at 10:25, Last updated: 8th Dec 2022 at 17:26

Central role of the RNA polymerase trigger loop in intrinsic RNA hydrolysis

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Nikolay Zenkin

Abstract (Expand)

The active center of RNA polymerase can hydrolyze phosphodiester bonds in nascent RNA, a reaction thought to be important for proofreading of transcription. The reaction proceeds via a general two … Mg(2+) mechanism and is assisted by the 3' end nucleotide of the transcript. Here, by using Thermus aquaticus RNA polymerase, we show that the reaction also requires the flexible domain of the active center, the trigger loop (TL). We show that the invariant histidine (beta' His1242) of the TL is essential for hydrolysis/proofreading and participates in the reaction in two distinct ways: by positioning the 3' end nucleotide of the transcript that assists catalysis and/or by directly participating in the reaction as a general base. We also show that participation of the beta' His1242 of the TL in phosphodiester bond hydrolysis does not depend on the extent of elongation complex backtracking. We obtained similar results with Escherichia coli RNA polymerase, indicating that the function of the TL in phosphodiester bond hydrolysis is conserved among bacteria.

Authors: Yulia Yuzenkova,

Date Published: 1st Jun 2010

Publication Type: Not specified

PubMed ID: 20534498

Citation:

Created: 17th Feb 2011 at 17:22, Last updated: 8th Dec 2022 at 17:26

Stepwise mechanism for transcription fidelity

Noisy-Strep

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Nikolay Zenkin

Yulia Yuzenkova

Abstract (Expand)

Transcription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just … non-complementary ribo NTP substrates but also against complementary 2'- and 3'-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive.

Authors: , Aleksandra Bochkareva, Vasisht R Tadigotla, Mohammad Roghanian, Savva Zorov, Konstantin Severinov,

Date Published: 1st Apr 2010

Publication Type: Not specified

PubMed ID: 20459653

Citation:

Created: 8th Feb 2011 at 11:52, Last updated: 8th Dec 2022 at 17:26

Gene position within a long transcript as a determinant for stochastic switching in bacteria

Noisy-Strep

Abstract (Expand)

How cultures of genetically identical cells bifurcate into distinct phenotypic subpopulations under uniform growth conditions is an important question in developmental biology of relevance even to … relatively simple developmental systems, such as spore formation in bacteria. A growing Bacillus subtilis culture consists of either cells that are motile and can swim or cells that are non-motile and are chained together. In this issue of Molecular Microbiology, Cozy and Kearns show that the probability of a cell to become motile depends on the position of the sigD gene within the long (27 kb) motility operon. sigD encodes the alternative sigma factor sigma(D) that, together with RNA polymerase, drives expression of genes required for cell separation and the assembly of flagella. sigD is the penultimate gene of the B. subtilis motility operon and, in the control strain approximately, 70% of the cells are motile. When sigD was moved upstream within the operon, a larger fraction of cells became motile (up to 100%). This study highlights that the position of a gene within an operon can have a large impact on the control of gene expression. Furthermore, it suggests that RNA polymerase processivity or mRNA turnover can play important roles as sources of noise in bacterial development, and that gene position might be an unrecognized and possibly widespread mechanism to regulate phenotypic variation.

Editor:

Date Published: 10th Mar 2010

Publication Type: Not specified

PubMed ID: 20233302

Citation:

Created: 8th Feb 2011 at 11:53, Last updated: 8th Dec 2022 at 17:26

Transformation of environmental Bacillus subtilis isolates by transiently inducing genetic competence

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Jan-Willem Veening

Abstract (Expand)

Domesticated laboratory strains of Bacillus subtilis readily take up and integrate exogenous DNA. In contrast, "wild" ancestors or Bacillus strains recently isolated from the environment can … only be genetically modified by phage transduction, electroporation or protoplast transformation. Such methods are laborious, have a variable yield or cannot efficiently be used to alter chromosomal DNA. A major disadvantage of using laboratory strains is that they have often lost, or do not display ecologically relevant physiologies such as the ability to form biofilms. Here we present a method that allows genetic transformation by natural competence in several environmental isolates of B. subtilis. Competence in these strains was established by expressing the B. subtilis competence transcription factor ComK from an IPTG-inducible promoter construct present on an unstable plasmid. This transiently activates expression of the genes required for DNA uptake and recombination in the host strain. After transformation, the comK encoding plasmid is lost easily because of its intrinsic instability and the transformed strain returns to its wild state. Using this method, we have successfully generated mutants and introduced foreign DNA into a number of environmental isolates and also B. subtilis strain NCIB3610, which is widely used to study biofilm formation. Application of the same method to strains of B. licheniformis was unsuccessful. The efficient and rapid approach described here may facilitate genetic studies in a wider array of environmental B. subtilis strains.

Authors: Reindert Nijland, J Grant Burgess, Jeff Errington,

Date Published: 11th Jan 2010

Publication Type: Not specified

PubMed ID: 20300532

Citation:

Created: 10th Feb 2011 at 10:26, Last updated: 8th Dec 2022 at 17:26

Significance analysis and statistical mechanics: an application to clustering

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Johannes Berg

Abstract (Expand)

This Letter addresses the statistical significance of structures in random data: given a set of vectors and a measure of mutual similarity, how likely is it that a subset of these vectors forms a cluster … with enhanced similarity among its elements? The computation of this cluster p value for randomly distributed vectors is mapped onto a well-defined problem of statistical mechanics. We solve this problem analytically, establishing a connection between the physics of quenched disorder and multiple-testing statistics in clustering and related problems. In an application to gene expression data, we find a remarkable link between the statistical significance of a cluster and the functional relationships between its genes.

Authors: Marta Łuksza, Michael Lässig,

Date Published: 27th Nov 2009

Publication Type: Not specified

PubMed ID: 21231375

Citation:

Created: 23rd Feb 2011 at 10:44, Last updated: 8th Dec 2022 at 17:26

Cellular localization of choline-utilization proteins in Streptococcus pneumoniae using novel fluorescent reporter systems

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Jan-Willem Veening

Abstract (Expand)

The molecular mechanisms underlying cell growth, cell division and pathogenesis in Streptococcus pneumoniae are still not fully understood. Single-cell methodologies are potentially of great value to … investigate S. pneumoniae cell biology. Here, we report the construction of novel plasmids for single and double cross-over integration of functional fusions to the gene encoding a fast folding variant of the green fluorescent protein (GFP) into the S. pneumoniae chromosome. We have also established a zinc-inducible system for the fine control of gfp-fusion gene expression and for protein depletion experiments in S. pneumoniae. Using this novel single cell toolkit, we have examined the cellular localization of the proteins involved in the essential process of choline decoration of S. pneumoniae teichoic acid. GFP fusions to LicA and LicC, enzymes involved in the activation of choline, showed a cytoplasmic distribution, as predicted from their primary sequences. A GFP fusion to the choline importer protein LicB showed clear membrane localization. GFP fusions to LicD1 and LicD2, enzymes responsible for loading of teichoic acid subunits with choline, are also membrane-associated, even though both proteins lack any obvious membrane spanning domain. These results indicate that the decoration of teichoic acid by the LicD enzymes is a membrane-associated process presumably occurring at lipid-linked teichoic acid precursors.

Authors: Alice Eberhardt, Ling J Wu, Jeff Errington, Waldemar Vollmer,

Date Published: 8th Sep 2009

Publication Type: Not specified

PubMed ID: 19737355

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Created: 10th Feb 2011 at 10:27, Last updated: 8th Dec 2022 at 17:26

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RNA-seq wild type Version 1

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Version 1 (earliest) Created 23rd Nov 2011 at 16:37 by Jan-Willem Veening

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