scholarly journals Escherichia coli mutations that prevent the action of the T4 unf/alc protein map in an RNA polymerase gene.

Genetics ◽  
1988 ◽  
Vol 118 (2) ◽  
pp. 173-180
Author(s):  
L Snyder ◽  
L Jorissen
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Strong Support ◽  
Rpob Gene ◽  
Late Gene Expression ◽  
Gene Encoding ◽  
E Coli ◽  
A Subunit ◽  
Phage Production

Abstract Bacteriophage T4 has the substituted base hydroxymethylcytosine in its DNA and presumably shuts off host transcription by specifically blocking transcription of cytosine-containing DNA. When T4 incorporates cytosine into its own DNA, the shutoff mechanism is directed back at T4, blocking its late gene expression and phage production. Mutations which permit T4 multiplication with cytosine DNA should be in genes required for host shutoff. The only such mutations characterized thus far have been in the phage unf/alc gene. The product of this gene is also required for the unfolding of the host nucleoid after infection, hence its dual name unf/alc. As part of our investigation of the mechanism of action of unf/alc, we have isolated Escherichia coli mutants which propagate cytosine T4 even if the phage are genotypically alc+. These same E. coli mutants are delayed in the T4-induced unfolding of their nucleoid, lending strong support to the conclusion that blocking transcription and unfolding the host nucleoid are but different manifestations of the same activity. We have mapped two of the mutations, called paf mutations for prevent alc function. They both map at about 90 min, probably in the rpoB gene encoding a subunit of RNA polymerase. From the behavior of Paf mutants, we hypothesize that the unf/alc gene product of T4 interacts somehow with the host RNA polymerase to block transcription of cytosine DNA and unfold the host nucleoid.

scholarly journals The Bacteriophage T4 Inhibitor and Coactivator AsiA Inhibits Escherichia coli RNA Polymerase More Rapidly in the Absence of σ70 Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ70 and Core

2006 ◽  
Vol 188 (4) ◽  
pp. 1279-1285 ◽  
Author(s):  
Deborah M. Hinton ◽  
Srilatha Vuthoori ◽  
Rebecca Mulamba
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Dynamic Equilibrium ◽  
Bacteriophage T4 ◽  
Direct Interaction ◽  
Binding Properties ◽  
E Coli ◽  
Dna Binding Properties ◽  
Two Hybrid

ABSTRACT The N-terminal region (region 1.1) of σ70, the primary σ subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of σ70 regions 2 and 4. Region 1.1 prevents the interaction of free σ70 with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of σ70-dependent transcription from promoters that require an interaction between σ70 region 4 and the −35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with σ70 region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that σ70 region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter P uvsX . However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with σ70 that lacks region 1.1 is less stable than polymerase with full-length σ70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free σ70 and then the AsiA/σ70 complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free σ70 plus core, yielding more free σ70 at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free σ70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free σ70 arises through an indirect effect.

scholarly journals A Mutation within the β Subunit of Escherichia coli RNA Polymerase Impairs Transcription from Bacteriophage T4 Middle Promoters

2010 ◽  
Vol 192 (21) ◽  
pp. 5580-5587 ◽  
Author(s):  
Tamara D. James ◽  
Michael Cashel ◽  
Deborah M. Hinton
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Β Subunit ◽  
Subunit Gene ◽  
Wild Type ◽  
Promoter Activation ◽  
E Coli ◽  
Growth Phenotype

ABSTRACT During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. Middle genes, whose expression begins about 1 min postinfection, are transcribed both from the extension of early RNA into middle genes and by the activation of T4 middle promoters. Middle-promoter activation requires the T4 transcriptional activator MotA and coactivator AsiA, which are known to interact with σ70, the specificity subunit of RNA polymerase. T4 motA amber [motA(Am)] or asiA(Am) phage grows poorly in wild-type E. coli. However, previous work has found that T4 motA(Am)does not grow in the E. coli mutant strain TabG. We show here that the RNA polymerase in TabG contains two mutations within its β-subunit gene: rpoB(E835K) and rpoB(G1249D). We find that the G1249D mutation is responsible for restricting the growth of either T4 motA(Am)or asiA(Am) and for impairing transcription from MotA/AsiA-activated middle promoters in vivo. With one exception, transcription from tested T4 early promoters is either unaffected or, in some cases, even increases, and there is no significant growth phenotype for the rpoB(E835K G1249D) strain in the absence of T4 infection. In reported structures of thermophilic RNA polymerase, the G1249 residue is located immediately adjacent to a hydrophobic pocket, called the switch 3 loop. This loop is thought to aid in the separation of the RNA from the DNA-RNA hybrid as RNA enters the RNA exit channel. Our results suggest that the presence of MotA and AsiA may impair the function of this loop or that this portion of the β subunit may influence interactions among MotA, AsiA, and RNA polymerase.

Upstream stimulators for recoding

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1123-1129 ◽  
Author(s):  
B. Larsen ◽  
K. Brady ◽  
J. F. Atkins ◽  
J. Peden ◽  
S. Matsufuji ◽  
...  
Keyword(s):  
Bacteriophage T4 ◽  
Gene Encoding ◽  
Dna Topoisomerase ◽  
E Coli ◽  
Optimal Spacing ◽  
A Subunit ◽  
Shine Dalgarno Sequence ◽  
Acid Domain ◽  
Frameshift Event ◽  
Amino Acid Domain

Recent progress in elucidation of 5′ stimulatory elements for translational recoding is reviewed. A 5′ Shine–Dalgarno sequence increases both +1 and −1 frameshift efficiency in several genes; examples cited include the E. coli prfB gene encoding release factor 2 and the dnaX gene encoding the γ and τ subunits of DNA polymerase III holoenzyme. The spacing between the Shine–Dalgarno sequence and the shift site is critical in both the +1 and −1 frameshift cassettes; however, the optimal spacing is quite different in the two cases. A frameshift in a mammalian chromosomal gene, ornithine decarboxylase antizyme, has recently been reported; 5′ sequences have been shown to be vital for this frameshift event. Escherichia coli bacteriophage T4 gene 60 encodes a subunit of its type II DNA topoisomerase. The mature gene 60 mRNA contains an internal 50 nucleotide region that appears to be bypassed during translation. A 16 amino acid domain of the nascent peptide is necessary for this bypass to occur.Key words: recoding, frameshifting, peptide factor, stimulatory elements.

scholarly journals Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

2008 ◽  
Vol 190 (10) ◽  
pp. 3434-3443 ◽  
Author(s):  
Umender K. Sharma ◽  
Dipankar Chatterji
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Sigma Factors ◽  
E Coli ◽  
Yeast Two Hybrid System ◽  
Vitro Binding

ABSTRACT Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, σ70, of E. coli. Though both factors are known to interact with the C-terminal region of σ70, the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to σ70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with σ70 studied by using the yeast two-hybrid system revealed that region 4 of σ70 is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of σ70 as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to σ70.

Mapping of sequences required for the translation of the β subunit of Escherichia coli RNA polymerase

1997 ◽  
Vol 43 (9) ◽  
pp. 819-826
Author(s):  
Luciano Passador ◽  
Thomas Linn
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Translational Regulation ◽  
Rpob Gene ◽  
Β Subunit ◽  
E Coli ◽  
Lacz Fusions ◽  
Translational Start ◽  
Genetic Constructs ◽  
Expression Plasmids

Previous experiments using expression plasmids which overproduce the β and β′ subunits of Escherichia coli RNA polymerase suggested that regions considerably upstream of the start of the rpoB gene, which encodes the β subunit, are required for its efficient synthesis. To further delineate the required regions, a collection of genetic constructs that contained varying amounts of the region either upstream or downstream of the translational start of rpoB was assembled. Measurements of β and β′ synthesis and rpoB mRNA production from a series of rpoBC expression plasmids indicated that sequences extending more than 43 bp but less than 79 bp upstream of rpoB are required for the efficient translation of rpoB mRNA. This result was confirmed by β-galactosidase measurements from a series of rpoB-lacZ fusions that have the same set of end points upstream of rpoB as the expression plasmids. A second set of gene fusions containing differing amounts of the sequence distal to the start of rpoB fused in frame to lacZ revealed that more than 29 bp but less than 70 bp of rpoB was required for efficient translation.Key words: RNA polymerase, E. coli, translational regulation.

scholarly journals In Vivo Transduction with Shiga Toxin 1-Encoding Phage

1998 ◽  
Vol 66 (9) ◽  
pp. 4496-4498 ◽  
Author(s):  
David W. K. Acheson ◽  
Joachim Reidl ◽  
Xiaoping Zhang ◽  
Gerald T. Keusch ◽  
John J. Mekalanos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gastrointestinal Tract ◽  
Shiga Toxin ◽  
Recipient Strain ◽  
Gene Encoding ◽  
E Coli ◽  
A Subunit

ABSTRACT To facilitate the study of intestinal transmission of the Shiga toxin 1 (Stx1)-converting phage H-19B, Tn10d-blamutagenesis of an Escherichia coli H-19B lysogen was undertaken. Two mutants containing insertions in the gene encoding the A subunit of Stx1 were isolated. The resultant ampicillin-resistantE. coli strains lysogenic for these phages produced infectious H-19B particles but not active toxin. These lysogens were capable of transducing an E. coli recipient strain in the murine gastrointestinal tract, thereby demonstrating that lysogens of Shiga toxin-converting phages give rise to infectious virions within the host gastrointestinal tract.

scholarly journals Growth Phase and Growth Rate Regulation of therapA Gene, Encoding the RNA Polymerase-Associated Protein RapA in Escherichia coli

2001 ◽  
Vol 183 (20) ◽  
pp. 6126-6134 ◽  
Author(s):  
Julio E. Cabrera ◽  
Ding Jun Jin
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Rna Polymerase ◽  
Growth Phase ◽  
Gene Encoding ◽  
Rate Regulation ◽  
E Coli ◽  
Growth Rate Control

ABSTRACT The Escherichia coli rapA gene encodes the RNA polymerase (RNAP)-associated protein RapA, which is a bacterial member of the SWI/SNF helicase-like protein family. We have studied therapA promoter and its regulation in vivo and determined the interaction between RNAP and the promoter in vitro. We have found that the expression of rapA is growth phase dependent, peaking at the early log phase. The growth phase control ofrapA is determined at least by one particular feature of the promoter: it uses CTP as the transcription-initiating nucleotide instead of a purine, which is used for most E. colipromoters. We also found that the rapA promoter is subject to growth rate regulation in vivo and that it forms intrinsic unstable initiation complexes with RNAP in vitro. Furthermore, we have shown that a GC-rich or discriminator sequence between the −10 and +1 positions of the rapA promoter is responsible for its growth rate control and the instability of its initiation complexes with RNAP.

scholarly journals DNA sequence around the Escherichia coli unc operon. Completion of the sequence of a 17 kilobase segment containing asnA, oriC, unc, glmS and phoS

1984 ◽  
Vol 224 (3) ◽  
pp. 799-815 ◽  
Author(s):  
J E Walker ◽  
N J Gay ◽  
M Saraste ◽  
A N Eberle
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Promoter Sequence ◽  
Gene Encoding ◽  
E Coli ◽  
Extensive Region ◽  
Transcriptional Promoters ◽  
Phosphoribosylpyrophosphate Amidotransferase ◽  
Unc Operon

The nucleotide sequence is described of a region of the Escherichia coli chromosome extending from oriC to phoS that also includes the loci gid, unc and glmS. Taken with known sequences for asnA and phoS this completes the sequence of a segment of about 17 kilobases or 0.4 min of the E. coli genome. Sequences that are probably transcriptional promoters for unc and phoS can be detected and the identity of the unc promoter has been confirmed by experiments in vitro with RNA polymerase. Upstream of the promoter sequence is an extensive region that appears to be non-coding. Conserved sequences are found that may serve to concentrate RNA polymerase in the vicinity of the unc promoter. Hairpin loop structures resembling known rho-independent transcription termination signals are evident following the unc operon and glmS. The glmS gene encoding the amidotransferase, glucosamine synthetase, has been identified by homology with glutamine 5-phosphoribosylpyrophosphate amidotransferase.

scholarly journals Pea chloroplast DNA encodes homologues of Escherichia coli ribosomal subunit S2 and the β'-subunit of RNA polymerase

1986 ◽  
Vol 236 (2) ◽  
pp. 453-460 ◽  
Author(s):  
A L Cozens ◽  
J E Walker
Keyword(s):  
Escherichia Coli ◽  
Nucleotide Sequence ◽  
Rna Polymerase ◽  
Atp Synthase ◽  
Ribosomal Subunit ◽  
Beta Subunit ◽  
Β Subunit ◽  
Membrane Domain ◽  
Gene Encoding ◽  
E Coli

The nucleotide sequence has been determined of a segment of 4680 bases of the pea chloroplast genome. It adjoins a sequence described elsewhere that encodes subunits of the F0 membrane domain of the ATP-synthase complex. The sequence contains a potential gene encoding a protein which is strongly related to the S2 polypeptide of Escherichia coli ribosomes. It also encodes an incomplete protein which contains segments that are homologous to the beta'-subunit of E. coli RNA polymerase and to yeast RNA polymerases II and III.

scholarly journals Transcriptional Activation of Agrobacterium tumefaciens Virulence Gene Promoters in Escherichia coli Requires the A. tumefaciens rpoA Gene, Encoding the Alpha Subunit of RNA Polymerase

1999 ◽  
Vol 181 (15) ◽  
pp. 4533-4539 ◽  
Author(s):  
S. M. Lohrke ◽  
S. Nechaev ◽  
H. Yang ◽  
K. Severinov ◽  
S. J. Jin
Keyword(s):  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Rna Polymerase ◽  
Virulence Genes ◽  
Virulence Gene ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Gene Encoding ◽  
Α Subunit ◽  
E Coli

ABSTRACT The two-component regulatory system, composed of virAand virG, is indispensable for transcription of virulence genes within Agrobacterium tumefaciens. However,virA and virG are insufficient to activate transcription from virulence gene promoters within Escherichia coli cells, indicating a requirement for additional A. tumefaciens genes. In a search for these additional genes, we have identified the rpoA gene, encoding the α subunit of RNA polymerase (RNAP), which confers significant expression of avirB promoter (virBp)::lacZ fusion in E. coli in the presence of an active transcriptional regulatorvirG gene. We conducted in vitro transcription assays using either reconstituted E. coli RNAP or hybrid RNAP in which the α subunit was derived from A. tumefaciens. The two forms of RNAP were equally efficient in transcription from a ς70-dependent E. coli galP1 promoter; however, only the hybrid RNAP was able to transcribe virBpin a virG-dependent manner. In addition, we provide evidence that the α subunit from A. tumefaciens, but not from E. coli, is able to interact with the VirG protein. These data suggest that transcription of virulence genes requires specific interaction between VirG and the α subunit of A. tumefaciens and that the α subunit from E. coli is unable to effectively interact with the VirG protein. This work provides the basis for future studies designed to examinevir gene expression as well as the T-DNA transfer process in E. coli.

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