Identifying the bicyclomycin binding domain through biochemical analysis of antibiotic-resistant rho proteins.
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Mutations M219K, S266A, and G337S in transcription termination factor Rho have been shown to confer resistance to the antibiotic bicyclomycin (BCM). All three His-tagged mutant Rho proteins exhibited similar Km values for ATP; however, the Vmax values at infinite ATP concentrations were one-fourth to one-third that for the His-tagged wild-type enzyme. BCM inhibition kinetics of poly(C)-dependent ATPase activity for the mutant proteins were non-competitive with respect to ATP (altering catalytic function but not ATP binding) and showed increased Ki values compared with His-tagged wild-type Rho. M219K and G337S exhibited increased ratios of poly(U)/poly(C)-stimulated ATPase activity and lower apparent Km values for ribo(C)10 in the poly(dC).ribo(C)10-dependent ATPase assay compared with His-tagged wild-type Rho. The S266A mutation did not show an increased poly(U)/poly(C) ATPase activity ratio and maintained approximately the same Km for ribo(C)10 in the poly(dC). ribo(C)10-dependent ATPase assay. The kinetic studies indicated that M219K and G337S altered the secondary RNA binding domain in Rho whereas the S266A mutation did not. Transcription termination assays for each mutant showed different patterns of Rho-terminated transcripts. Tyrosine substitution of Ser-266 led to BCM sensitivity intimating that an OH (hydroxyl) moiety at this position is needed for BCM (binding) inhibition. Our results suggest BCM binds to Rho at a site distinct from both the ATP and the primary RNA binding domains but close to the secondary RNA-binding (tracking) site and the ATP hydrolysis pocket. (+info)
Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157:H7: Shiga toxin as a phage late-gene product.
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Lysogenic bacteriophages are major vehicles for the transfer of genetic information between bacteria, including pathogenicity and/or virulence determinants. In the enteric pathogen Escherichia coli O157:H7, which causes hemorrhagic colitis and hemolytic-uremic syndrome, Shiga toxins 1 and 2 (Stx1 and Stx2) are phage encoded. The sequence and analysis of the Stx2 phage 933W is presented here. We find evidence that the toxin genes are part of a late-phage transcript, suggesting that toxin production may be coupled with, if not dependent upon, phage release during lytic growth. Another phage gene, stk, encodes a product resembling eukaryotic serine/threonine protein kinases. Based on its position in the sequence, Stk may be produced by the prophage in the lysogenic state, and, like the YpkA protein of Yersinia species, it may interfere with the signal transduction pathway of the mammalian host. Three novel tRNA genes present in the phage genome may serve to increase the availability of rare tRNA species associated with efficient expression of pathogenicity determinants: both the Shiga toxin and serine/threonine kinase genes contain rare isoleucine and arginine codons. 933W also has homology to lom, encoding a member of a family of outer membrane proteins associated with virulence by conferring the ability to survive in macrophages, and bor, implicated in serum resistance. (+info)
Dye structure affects Taq DNA polymerase terminator selectivity.
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All DNA sequencing methods have benefited from the development of new F667Y versions of Taq DNA polymerase. However, terminator chemistry methods show less uniform peak height patterns when compared to primer chemistry profiles suggesting that the dyes and/or their linker arms affect enzyme selectivity. We have measured elementary nucleotide rate and binding constants for representative rhodamine- and fluorescein-labeled terminators to determine how they interact with F667 versions of Taq Pol I. We have also developed a rapid gel-based selectivity assay that can be used to screen and to quantify dye-enzyme interactions with F667Y versions of the enzyme. Our results show that 6-TAMRA-ddTTP behaves like unlabeled ddTTP, while 6-FAM-ddTTP shows a 40-fold reduction in the rate constant for polymerization without affecting ground-state nucleotide binding. Detailed mechanism studies indicate that both isomers of different fluorescein dyes interfere with a conformational change step which the polymerase undergoes following nucleotide binding but only when these dyes are attached to pyrimidines. When these same dyes are attached to purines by the same propargylamino linker arm, they show no effect on enzyme selectivity. These studies suggest that it may be possible to develop fluorescein terminators for thermocycle DNA sequencing methods for polymerases that do not discriminate between deoxy- and dideoxynucleotides. (+info)
Analysis of an autoregulatory loop controlling ToxT, cholera toxin, and toxin-coregulated pilus production in Vibrio cholerae.
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Coordinate expression of many virulence genes in the human pathogen Vibrio cholerae is controlled by the ToxR, TcpP, and ToxT proteins. These proteins function in a regulatory cascade in which ToxR and TcpP, two inner membrane proteins, are required to activate toxT and ToxT is the direct activator of virulence gene expression. ToxT-activated genes include those whose products are required for the biogenesis of cholera toxin (CTX) and the toxin-coregulated pilus, the major subunit of which is TcpA. This work examined control of toxT transcription. We tested a model whereby activation of toxT by ToxR and TcpP is required to prime an autoregulatory loop in which ToxT-dependent transcription of the tcpA promoter reads through a proposed terminator between the tcpF and toxT genes to result in continued ToxT production. Primer extension analysis of RNA from wild-type classical strain O395 showed that there are two products encoding toxT, one of which is longer than the other by 105 bp. Deletion of the toxT promoter (toxTDeltapro) resulted in the abolishment of toxT transcription, as predicted. Deletion of the tcpA promoter (tcpADeltapro) had no effect on subsequent detection of the smaller toxT primer extension product, but the larger toxT product was not detected, indicating that this product may be the result of transcription from the tcpA promoter and not of initiation directly upstream of toxT. Neither mutant strain produced detectable TcpA, but the CTX levels of the strains were different. The toxTDeltapro strain produced little detectable CTX, while the tcpADeltapro strain produced CTX levels intermediate between those of the wild-type and toxTDeltapro strains. Dependence of toxT transcription on TcpP and TcpH was confirmed by analyzing RNAs from strains carrying deletions in the genes encoding these regulators. The tcpP defect resulted in undetectable toxT transcription, whereas the tcpH mutation led to a diminishing of toxT RNA but not complete abolishment. Taken together, these results suggest that toxT transcription is dependent on two different promoters; one is directly upstream and is activated in part by TcpP and TcpH, and the other is much further upstream and is activated by ToxT. (+info)
Mechanism of intrinsic transcription termination and antitermination.
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Gene expression is modulated by regulatory elements that influence transcription elongation by RNA polymerase: terminators that disrupt the elongation complex and release RNA, and regulators that overcome termination signals. RNA release from Escherichia coli RNA polymerase can be induced by a complementary oligonucleotide that replaces the upstream half of the RNA hairpin stem of intrinsic terminator transcripts, implying that RNA hairpins act by extracting RNA from the transcription complex. A transcription antiterminator inhibits this activity of oligonucleotides and therefore protects the elongation complex from destabilizing attacks on the emerging transcript. These effects illuminate the structure of the complex and the mechanism of transcription termination. (+info)
Modulation of epidermal growth factor receptor gene transcription by a polymorphic dinucleotide repeat in intron 1.
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The influence of a highly polymorphic CA dinucleotide repeat in the epidermal growth factor receptor (EGFR) gene on transcription was examined with a quantitative nuclear run-off method. We could demonstrate that transcription of the EGFR gene is inhibited by approximately 80% in alleles with 21 CA repeats. In experiments with polymerase chain reaction products that spanned a region of more than 4,000 base pairs and contained the promoter, two enhancers, and the polymorphic region in the first intron of the gene, we found that transcription activity declines with increasing numbers of CA dinucleotides. In vivo pre-mRNA expression data from cultured cell lines support these findings, although other regulation mechanisms can outweigh this effect. In addition, we showed that under our experimental conditions RNA elongation terminates at a site closely downstream of the simple sequence repeat and that there are two separate major transcription start sites. Our results provide new insights in individually different EGFR gene expression and the role of the CA repeat in transcription of this proto-oncogene. (+info)
Catabolite control of Escherichia coli regulatory protein BglG activity by antagonistically acting phosphorylations.
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In bacteria various sugars are taken up and concomitantly phosphorylated by sugar-specific enzymes II (EII) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphoryl groups are donated by the phosphocarrier protein HPr. BglG, the positively acting regulatory protein of the Escherichia coli bgl (beta-glucoside utilization) operon, is known to be negatively regulated by reversible phosphorylation catalyzed by the membrane spanning beta-glucoside-specific EIIBgl. Here we present evidence that in addition BglG must be phosphorylated by HPr at a distinct site to gain activity. Our data suggest that this second, shortcut route of phosphorylation is used to monitor the state of the various PTS sugar availabilities in order to hierarchically tune expression of the bgl operon in a physiologically meaningful way. Thus, the PTS may represent a highly integrated signal transduction network in carbon catabolite control. (+info)
mRNA surveillance in eukaryotes: kinetic proofreading of proper translation termination as assessed by mRNP domain organization?
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In the last few years it has become clear that a conserved mRNA degradation system, referred to as mRNA surveillance, exists in eukaryotic cells to degrade aberrant mRNAs. This process plays an important role in checking that mRNAs have been properly synthesized and functions, at least in part, to increase the fidelity of gene expression by degrading aberrant mRNAs that, if translated, would produce truncated proteins. A critical issue is how normal and aberrant mRNAs are distinguished and how that distinction leads to differences in mRNA stability. Recent results suggest a model with three main points. First, mRNPs have a domain organization that is, in part, a reflection of the completion of nuclear pre-mRNA processing events. Second, the critical aspect of distinguishing a normal from an aberrant mRNA is the environment of the translation termination codon as determined by the organization of the mRNP domains. Third, the cell distinguishes proper from improper termination through an internal clock that is the rate of ATP hydrolysis by Upf1p. If termination is completed before ATP hydrolysis, the mRNA is protected from mRNA degradation. Conversely, if termination is slow, then ATP hydrolysis and a structural rearrangement occurs before termination is completed, which affects the fate of the terminating ribosome in a manner that fails to stabilize the mRNA. This proposed system of distinguishing normal from aberrant transcripts is similar to, but distinct from other systems of kinetic proofreading that affect the accuracy of other biogenic processes such as translation accuracy and spliceosome assembly. (+info)