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Find video protocols related to scientific articles indexed in Pubmed.
Expression of each cistron in the gal operon can be regulated by transcription termination and generation of a galk-specific mRNA, mK2.
J. Bacteriol.
PUBLISHED: 05-02-2014
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The gal operon of Escherichia coli has 4 cistrons, galE, galT, galK, and galM. In our previous report (H. J. Lee, H. J. Jeon, S. C. Ji, S. H. Yun, H. M. Lim, J. Mol. Biol. 378: 318-327, 2008), we identified 6 different mRNA species, mE1, mE2, mT1, mK1, mK2, and mM1, in the gal operon and mapped these mRNAs. The mRNA map suggests a gradient of gene expression known as natural polarity. In this study, we investigated how the mRNAs are generated to understand the cause of natural polarity. Results indicated that mE1, mT1, mK1, and mM1, whose 3' ends are located at the end of each cistron, are generated by transcription termination. Since each transcription termination is operating with a certain frequency and those 4 mRNAs have 5' ends at the transcription initiation site(s), these transcription terminations are the basic cause of natural polarity. Transcription terminations at galE-galT and galT-galK junctions, making mE1 and mT1, are Rho dependent. However, the terminations to make mK1 and mM1 are partially Rho dependent. The 5' ends of mK2 are generated by an endonucleolytic cleavage of a pre-mK2 by RNase P, and the 3' ends are generated by Rho termination 260 nucleotides before the end of the operon. The 5' portion of pre-mK2 is likely to become mE2. These results also suggested that galK expression could be regulated through mK2 production independent from natural polarity.
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In vivo transcription dynamics of the galactose operon: a study on the promoter transition from P1 to P2 at onset of stationary phase.
PLoS ONE
PUBLISHED: 02-09-2011
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Quantitative analyses of the 5 end of gal transcripts indicate that transcription from the galactose operon P1 promoter is higher during cell division. When cells are no longer dividing, however, transcription is initiated more often from the P2 promoter. Escherichia coli cells divide six times before the onset of the stationary phase when grown in LB containing 0.5% galactose at 37°C. Transcription from the two promoters increases, although at different rates, during early exponential phase (until the third cell division, OD(600) 0.4), and then reaches a plateau. The steady-state transcription from P1 continues in late exponential phase (the next three cell divisions, OD(600) 3.0), after which transcription from this promoter decreases. However, steady-state transcription from P2 continues 1 h longer into the stationary phase, before decreasing. This longer steady-state P2 transcription constitutes the promoter transition from P1 to P2 at the onset of the stationary phase. The intracellular cAMP concentration dictates P1 transcription dynamics; therefore, promoter transition may result from a lack of cAMP-CRP complex binding to the gal operon. The decay rate of gal-specific transcripts is constant through the six consecutive cell divisions that comprise the exponential growth phase, increases at the onset of the stationary phase, and is too low to be measured during the stationary phase. These data suggest that a regulatory mechanism coordinates the synthesis and decay of gal mRNAs to maintain the observed gal transcription. Our analysis indicates that the increase in P1 transcription is the result of cAMP-CRP binding to increasing numbers of galactose operons in the cell population.
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Quantification of the galactose-operon mRNAs 5 bases different in their 5-ends.
BMB Rep
PUBLISHED: 07-29-2010
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Three assay methods for quantification of the two galactoseoperon mRNAs that only differ by 5 bases in their 5-end are presented. The 5 ends of each mRNA were extended by ligating the 3-end of the abundant 5S rRNA. This ligation extends the 5 ends of the two gal mRNAs long enough to be distinguished by the specific PCR primers in the following quantification reactions. Quantification of the corresponding cDNAs was performed either by primer extension assay or real-time qPCR. To circumvent the problem of the RNA ligation reaction (i.e. very low ligation efficiency), we devised a new method that employs real-time qPCR directly for the quantification of the gal transcripts which differ by 5 bases in their 5-ends.
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A mutational study of Cnu reveals attractive forces between Cnu and H-NS.
Mol. Cells
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Cnu is a small 71-amino acid protein that complexes with H-NS and binds to a specific sequence in the replication origin of the E. coli chromosome. To understand the mechanism of interaction between Cnu and H-NS, we used bacterial genetics to select and analyze Cnu variants that cannot complex with H-NS. Out of 2,000 colonies, 40 Cnu variants were identified. Most variants (82.5%) had a single mutation, but a few variants (17.5%) had double amino acid changes. An in vitro assay was used to identify Cnu variants that were truly defective in H-NS binding. The changes in these defective variants occurred exclusively at charged amino acids (Asp, Glu, or Lys) on the surface of the protein. We propose that the attractive force that governs the Cnu-H-NS interaction is an ionic bond, unlike the hydrophobic interaction that is the major attractive force in most proteins.
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The CnuK9E H-NS complex antagonizes DNA binding of DicA and leads to temperature-dependent filamentous growth in E. coli.
PLoS ONE
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Cnu (an OriC-binding nucleoid protein) associates with H-NS. A variant of Cnu was identified as a key factor for filamentous growth of a wild-type Escherichia coli strain at 37°C. This variant (CnuK9E) bears a substitution of a lysine to glutamic acid, causing a charge reversal in the first helix. The temperature-dependent filamentous growth of E. coli bearing CnuK9E could be reversed by either lowering the temperature to 25°C or lowering the CnuK9E concentration in the cell. Gene expression analysis suggested that downregulation of dicA by CnuK9E causes a burst of dicB transcription, which, in turn, elicits filamentous growth. In vivo assays indicated that DicA transcriptionally activates its own gene, by binding to its operator in a temperature-dependent manner. The antagonizing effect of CnuK9E with H-NS on DNA-binding activity of DicA was stronger at 37°C, presumably due to the lower operator binding of DicA at 37°C. These data suggest that the temperature-dependent negative effect of CnuK9E on DicA binding plays a major role in filamentous growth. The C-terminus of DicA shows significant amino acid sequence similarity to the DNA-binding domains of RovA and SlyA, regulators of pathogenic genes in Yersinia and Salmonella, respectively, which also show better DNA-binding activity at 25°C.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.