Home About us Contact
|
Home About us Contact | ||
|
|
||
Dependent Promoter (dependent + promoter)
Selected AbstractsThe expression of recombinant genes in Escherichia coli can be strongly stimulated at the transcript production level by mutating the DNA-region corresponding to the 5,-untranslated part of mRNAMICROBIAL BIOTECHNOLOGY, Issue 3 2009Laila Berg Summary Secondary structures and the short Shine,Dalgarno sequence in the 5,-untranslated region of bacterial mRNAs (UTR) are known to affect gene expression at the level of translation. Here we report the use of random combinatorial DNA sequence libraries to study UTR function, applying the strong, ,32/,38 -dependent, and positively regulated Pm promoter as a model. All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site. The libraries were screened using the ampicillin-resistance gene (bla) as reporter, allowing easy identification of UTR mutants that display high levels of expression (up to 20-fold increase relative to the wild-type at the protein level). Studies of the two UTR mutants identified by a modified screening procedure showed that their expression is stimulated to a similar extent at both the transcript and protein product levels. For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18-fold higher than the wild type), while there was no evidence of a difference in transcript stability. The two UTR sequences also stimulated expression from a constitutive ,70 -dependent promoter (P1/Panti-tet), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription. [source] Gene position in a long operon governs motility development in Bacillus subtilisMOLECULAR MICROBIOLOGY, Issue 2 2010Loralyn M. Cozy Summary Growing cultures of Bacillus subtilis bifurcate into subpopulations of motile individuals and non-motile chains of cells that are differentiated at the level of gene expression. The motile cells are ON and the chaining cells are OFF for transcription that depends on RNA polymerase and the alternative sigma factor ,D. Here we show that chaining cells were OFF for ,D -dependent gene expression because ,D levels fell below a threshold and ,D activity was inhibited by the anti-sigma factor FlgM. The probability that ,D exceeded the threshold was governed by the position of the sigD gene. The proportion of ON cells increased when sigD was artificially moved forward in the 27 kb fla/che operon. In addition, we identified a new ,D -dependent promoter that increases sigD expression and may provide positive feedback to stabilize the ON state. Finally, we demonstrate that ON/OFF motility states in B. subtilis are a form of development because mosaics of stable and differentiated epigenotypes were evident when the normally dispersed bacteria were forced to grow in one dimension. [source] A novel NO-responding regulator controls the reduction of nitric oxide in Ralstonia eutrophaMOLECULAR MICROBIOLOGY, Issue 3 2000Anne Pohlmann Ralstonia eutropha H16 mediates the reduction of nitric oxide (NO) to nitrous oxide (N2O) with two isofunctional single component membrane-bound NO reductases (NorB1 and NorB2). This reaction is integrated into the denitrification pathway that involves the successive reduction of nitrate to dinitrogen. The norB1 gene is co-transcribed with norA1 from a ,54 (RpoN)-dependent promoter, located upstream of norA1. With the aid of norA1,,lacZ transcriptional fusions and the generation of regulatory mutants, it was shown that norB1 gene transcription requires a functional rpoN gene and the regulator NorR, a novel member of the NtrC family of response regulators. The regulator gene maps adjacent to norAB, is divergently transcribed and present in two copies on the megaplasmid pHG1 (norR1) and the chromosome (norR2). Transcription activation by NorR responds to the availability of NO. A nitrite reductase-deficient mutant that is incapable of producing NO endogenously, showed a 70% decrease of norA1 expression. Addition of the NO-donating agent sodium nitroprusside caused induction of norA1,,lacZ transcription. Truncation of the N-terminal receiver domain of NorR1 interrupted the NO signal transduction and led to a constitutive expression of norA1,,lacZ. The results indicate that NorR controls the reductive conversion of NO in R. eutropha. This reaction is not strictly co-ordinated on the regulatory level with the other nitrogen oxide-reducing steps of the denitrification chain that are independent of NorR. [source] Changes in lipopolysaccharide structure induce the ,E -dependent response of Escherichia coliMOLECULAR MICROBIOLOGY, Issue 5 2005Christina Tam Summary The envelope of Escherichia coli is composed of an asymmetric lipid bilayer containing lipopolysaccharide, phospholipid and outer membrane proteins (OMPs). Physical and chemical stresses impact on the integrity of the outer membrane envelope and trigger the ,E -dependent response, whereby E. coli activates the expression of genes that increase its capacity for folding OMPs and synthesizing lipopolysaccharide (LPS). While it has already been appreciated that misfolded OMPs induce the ,E response, a role for LPS in activating this pathway was hitherto unknown. Here we show that ammonium metavandate (NH4VO3) induces multiple changes in E. coli LPS structure and activates the ,E -dependent response without altering OMP. One such NH4VO3 -mediated LPS decoration, the CrcA/PagP-catalysed addition of palmitate to lipid A, appeared to be alone sufficient to activate transcription at ,E -dependent promoters. Furthermore, reduced acylation of LPS, caused by htrB or msbB mutations, also resulted in a constitutive expression of the ,E regulon above wild-type levels. Production of these aberrant outer membrane lipids did not noticeably affect the composition or the amount of OMPs. A model is proposed whereby structural intermediates of the LPS biosynthetic pathway or modified LPS molecules may function as signals that activate the ,E response. [source] | ||||||||||