scholarly journals Correlation between the 32-kDa sigma factor levels and in vitro expression of Escherichia coli heat shock genes.

1987 ◽  
Vol 84 (23) ◽  
pp. 8365-8369 ◽  
Author(s):  
S. Skelly ◽  
T. Coleman ◽  
C. F. Fu ◽  
N. Brot ◽  
H. Weissbach
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Sigma Factor ◽  
Heat Shock Genes ◽  
In Vitro Expression

scholarly journals Substitution of a Highly Conserved Histidine in the Escherichia coli Heat Shock Transcription Factor, σ32, Affects Promoter Utilization In Vitro and Leads to Overexpression of the Biofilm-Associated Flu Protein In Vivo

2007 ◽  
Vol 189 (23) ◽  
pp. 8430-8436 ◽  
Author(s):  
Olga V. Kourennaia ◽  
Pieter L. deHaseth
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Stable Complex ◽  
Tryptophan Residue ◽  
Specific Sequence ◽  
Bacterial Rna

ABSTRACT The heat shock sigma factor (σ32 in Escherichia coli) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of σ32 is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of E. coli, σ70. This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing σ32, implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in σ32 impedes recovery from heat shock (exposure to 42°C), and it also leads to overexpression at lower temperatures (30°C) of the Flu protein, which is associated with biofilm formation.

The Escherichia coli chaperones involved in DNA replicadon

1993 ◽  
Vol 339 (1289) ◽  
pp. 271-278 ◽  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Conformational Changes ◽  
Heat Shock Genes ◽  
Dnab Helicase ◽  
Initiation Of Dna Replication ◽  
Shock Proteins

Mutadons in the Escherichia coli heat shock genes, dnaK , dnaJ or grpE , alter host DNA and RNA synthesis, degradation of other proteins, cell division and expression of other heat shock genes. They also block the initiation of DNA replication of bacteriophages λ and P1, and the mini-F plasmid. An in vitro λDNA replication system, composed entirely of purified components, enabled us to describe the molecular mechanism of the dnaK , dnaJ and grpE gene products. DnaK , the bacterial hsp 70 homologue, releases λP protein from the preprimosomal complex in an ATP- and DnaJ-dependent reaction (GrpEindependent initiation of λDNA replication). In this paper, I show that, when GrpE is present, λP protein is not released from the preprimosomal complex, rather it is translocated within the complex in such a way that it does not inhibit DnaB helicase activity. Translocation of λP triggers the initiation event allowing DnaB helicase to unwind DNA near the ori λ sequence, leading to efficient λDNA replication. Chaperone activity of the DnaK -DnaJ-GrpE system is first manifested in the selective binding of these heat shock proteins to the preprimosomal complex, followed by its ATP-dependent rearrangement. I show that DnaJ not only tags the preprimosomal complex for recognition by DnaK, but also stabilizes the multi-protein structure. GrpE also participates in the binding of DnaK to the preprimosomal complex by increasing DnaK ’s affinity to those λP proteins which are already associated with DnaJ. After attracting DnaK to the preprimosomal complex, DnaJ and GrpE stimulate the ATPase activity of DnaK , triggering conformational changes in DnaK which are responsible for the rearrangement of proteins in the preprimosomal complex and recycling of these heat shock proteins. The role of DnaK , DnaJ and GrpE in λDNA replication is in sharp contrast to our understanding of their role in the oriC , P1, and probably mini-F DNA replication systems. In the cases of oriC and P1 DNA replication, these heat shock proteins activate initiation factors before they are in contact with DNA, and are not required during the subsequent steps leading to the initiation of DNA replication. The common feature of DnaK , DnaJ and GrpE action in these systems is their ATP-dependent disaggregation or rearrangement of protein complexes formed before or during initiation of DNA replication.

scholarly journals In vitro effect of the Escherichia coli heat shock regulatory protein on expression of heat shock genes.

1986 ◽  
Vol 166 (2) ◽  
pp. 380-384 ◽  
Author(s):  
M Bloom ◽  
S Skelly ◽  
R VanBogelen ◽  
F Neidhardt ◽  
N Brot ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Regulatory Protein ◽  
Heat Shock Genes ◽  
Vitro Effect

Cloning the gene for the heat shock response positiwe regulator (sigma 32 homolog) from Pseudomonas aeruginosa

1995 ◽  
Vol 41 (1) ◽  
pp. 75-87 ◽  
Author(s):  
Zerlina M. Naczynski ◽  
Andrew M. Kropinski ◽  
Chris Mueller
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Heat Shock ◽  
Sigma Factor ◽  
Oligonucleotide Probe ◽  
In Vitro Transcription ◽  
Translation System ◽  
Amino Acid Homology ◽  
E Coli ◽  
Transcriptional Start Sites

A 31 base pair synthetic oligonucleotide based on the genes for the Escherichia coli heat shock sigma factor (rpoH) and the Pseudomonas aeruginosa housekeeping sigma factor (rpoD) was employed in conjunction with the Tanaka et al. (K. Tanaka, T. Shiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040–1042) RpoD box probe to identify the location of the rpoH gene in P. aeruginosa genomic digests. This gene was cloned into plasmid pGEM3Z(f+), sequenced, and found to share 67% nucleotide identity and 77% amino acid homology with the rpoH gene and its product (σ32) of E. coli. The plasmid containing the rpoH gene complemented the function of σ32 in an E. coli rpoH deletion mutant. Furthermore, this plasmid directed the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transcription–translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50 °C) conditions. Two promoter sites were identified having sequence homology to the E. coli σ70 and σ24 consensus sequences.Key words: heat shock, Pseudomonas aeruginosa, sigma factor, transcription, oligonucleotide probe.

In vitro expression of Escherichia coli ribosomal protein L10 gene: Tripeptide synthesis as a measure of functional mRNA

1982 ◽  
Vol 218 (2) ◽  
pp. 572-578 ◽  
Author(s):  
Yves Cenatiempo ◽  
Nikolaos Robakis ◽  
Brian R. Reid ◽  
Herbert Weissbach ◽  
Nathan Brot
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Protein ◽  
In Vitro Expression

scholarly journals Transcriptome and translatome changes in germinated pollen under heat stress uncover roles of transporter genes involved in pollen tube growth

2020 ◽  
Author(s):  
Laetitia Poidevin ◽  
Javier Forment ◽  
Dilek Unal ◽  
Alejandro Ferrando
Keyword(s):  
Arabidopsis Thaliana ◽  
Heat Stress ◽  
High Temperature ◽  
Heat Shock ◽  
Pollen Tube ◽  
Pollen Tube Growth ◽  
Tube Growth ◽  
Heat Shock Genes ◽  
Germinated Pollen

ABSTRACTPlant reproduction is one key biological process very sensitive to heat stress and, as a consequence, enhanced global warming poses serious threats to food security worldwide. In this work we have used a high-resolution ribosome profiling technology to study how heat affects both the transcriptome and the translatome of Arabidopsis thaliana pollen germinated in vitro. Overall, a high correlation between transcriptional and translational responses to high temperature was found, but specific regulations at the translational level were also present. We show that bona fide heat shock genes are induced by high temperature indicating that in vitro germinated pollen is a suitable system to understand the molecular basis of heat responses. Concurrently heat induced significant down-regulation of key membrane transporters required for pollen tube growth, thus uncovering heat-sensitive targets. We also found that a large subset of the heat-repressed transporters is specifically up-regulated, in a coordinated manner, with canonical heat-shock genes in pollen tubes grown in vitro and semi in vivo, based on published transcriptomes from Arabidopsis thaliana. Ribosome footprints were also detected in gene sequences annotated as non-coding, highlighting the potential for novel translatable genes and translational dynamics.

scholarly journals The Skn7 Response Regulator ofSaccharomyces cerevisiaeInteracts with Hsf1 In Vivo and Is Required for the Induction of Heat Shock Genes by Oxidative Stress

2000 ◽  
Vol 11 (7) ◽  
pp. 2335-2347 ◽  
Author(s):  
Desmond C. Raitt ◽  
Anthony L. Johnson ◽  
Alexander M. Erkine ◽  
Kozo Makino ◽  
Brian Morgan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Response Regulator ◽  
Coiled Coil ◽  
Normal Growth ◽  
Temperature Sensitive ◽  
Regulatory Sequences ◽  
Heat Shock Genes

The Skn7 response regulator has previously been shown to play a role in the induction of stress-responsive genes in yeast, e.g., in the induction of the thioredoxin gene in response to hydrogen peroxide. The yeast Heat Shock Factor, Hsf1, is central to the induction of another set of stress-inducible genes, namely the heat shock genes. These two regulatory trans-activators, Hsf1 and Skn7, share certain structural homologies, particularly in their DNA-binding domains and the presence of adjacent regions of coiled-coil structure, which are known to mediate protein–protein interactions. Here, we provide evidence that Hsf1 and Skn7 interact in vitro and in vivo and we show that Skn7 can bind to the same regulatory sequences as Hsf1, namely heat shock elements. Furthermore, we demonstrate that a strain deleted for the SKN7 gene and containing a temperature-sensitive mutation in Hsf1 is hypersensitive to oxidative stress. Our data suggest that Skn7 and Hsf1 cooperate to achieve maximal induction of heat shock genes in response specifically to oxidative stress. We further show that, like Hsf1, Skn7 can interact with itself and is localized to the nucleus under normal growth conditions as well as during oxidative stress.

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