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Transcript Stability (transcript + stability)
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] Cold-shock-induced de novo transcription and translation of infA and role of IF1 during cold adaptationMOLECULAR MICROBIOLOGY, Issue 3 2007Mara Giangrossi Summary Escherichia coli infA is transcribed from two promoters, P1 and P2, into a longer and a shorter mRNA encoding translation initiation factor IF1. Although P1 is intrinsically stronger than P2, the shorter half-life of its transcripts causes the steady-state level of the P2 transcript to be substantially higher than that of P1 during growth at 37°C. After cold-shock, de novo transcription and translation of infA contribute to the transient increase of the IF1/ribosomes ratio, which is partially responsible for translational bias consisting in the preferential translation of cold-shock mRNAs in the cold. Cold-stress induction of infA expression is mainly due to the high activity of P1 at low temperature, which is further increased by transcriptional stimulation by CspA and by an increased transcript stability. Furthermore, the longer infA mRNA originating from P1 is preferentially translated at low temperature by the translational machinery of cold-shocked cells. The increased level of IF1 during cold adaptation is essential for overcoming the higher stability of the 70S monomers at low temperature and for providing a sufficient pool of dissociated 30S subunits capable of initiating translation. [source] Polyadenylation of Escherichia coli transcripts plays an integral role in regulating intracellular levels of polynucleotide phosphorylase and RNase EMOLECULAR MICROBIOLOGY, Issue 5 2002Bijoy K. Mohanty Summary Polyadenylation in Escherichia coli has been implicated in the destabilization of a variety of transcripts. However, transiently increasing intracellular poly(A) levels has also been shown to stabilize the pnp and rne transcripts, leading to increased polynucleotide phosphorylase (PNPase) and RNase E levels respectively. Here, we show that the half-lives of both the pnp and rne transcripts are dependent on the intracellular level of polyadenylated transcripts. In addition, experiments using pnp,lacZ and rne,lacZ translational fusions demonstrate that the variations in transcript stability and protein levels arise from alterations in the autoregulation of both genes. Further support for this conclusion is provided by the fact that, in an rne mutant in which autoregulation is inactivated by deletion of most of the 5, untranslated region, variations in the level of polyadenylated transcripts no longer affect RNase E protein expression. Of even more interest is the fact that the presence of a functional degradosome is essential for RNase E to detect increased levels of poly(A). Thus, it appears that polyadenylation of transcripts in E. coli serves as a sensing mechanism by which the cell adjusts the levels of both RNase E and PNPase. [source] SAG2 and SAG12 protein expression in senescing Arabidopsis plantsPHYSIOLOGIA PLANTARUM, Issue 2 2003Vojislava Grbi During leaf senescence, nutrients are remobilized from the senescing tissues to the growing parts of the plant. Many senescence-associated genes (SAGs) were identified based on the induction of their transcripts. However, little is known about the protein expression of the corresponding genes. We have raised antibodies against two Arabidopsis SAGs, SAG2 and SAG12, which encode putative cysteine proteases. The SAG2 antibodies recognized a 29-kDa protein that was abundant in senescing leaves, but was also present at low levels in green tissues. SAG12 antibodies labelled a 38-kDa protein present only in senescent leaves. The protein expression of these SAGs parallels their mRNA expression patterns, indicating that control of SAG2 and SAG12 is at the level of transcription or transcript stability. In addition, we found that SAGs are induced during stem senescence with delayed kinetics of their expression relative to leaf expression, suggesting that age-dependent factor(s) regulating the onset of senescence in Arabidopsis may act in tissue-dependent manner. [source] | ||||||||||