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Published year: 20105
DnaA and ORC: more than DNA replication initiators

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

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

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

Stepwise mechanism for transcription fidelity

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

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

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

Transformation of environmental Bacillus subtilis isolates by transiently inducing genetic competence

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

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