Designing sequential transcription logic: a simple genetic circuit for conditional memory

The ability to learn and respond to recurrent events depends on the capacity to remember transient biological signals received in the past. Moreover, it may be desirable to remember or ignore these transient signals conditioned upon other signals that are active at specific points in time or in unique environments. Here, we propose a simple genetic circuit in bacteria that is capable of conditionally memorizing a signal in the form of a transcription factor concentration. The circuit behaves similarly to a "data latch" in an electronic circuit, i.e. it reads and stores an input signal only when conditioned to do so by a "read command". Our circuit is of the same size as the well-known genetic toggle switch (an unconditional latch) which consists of two mutually repressing genes, but is complemented with a "regulatory front end" involving protein heterodimerization as a simple way to implement conditional control. Deterministic and stochastic analysis of the circuit dynamics indicate that an experimental implementation is feasible based on well-characterized genes and proteins. It is not known, to which extent molecular networks are able to conditionally store information in natural contexts for bacteria. However, our results suggest that such sequential logic elements may be readily implemented by cells through the combination of existing protein-protein interactions and simple transcriptional regulation.Comment: 20 pages, 5 figures; supplementary material available upon request from the author

Global Systems-Level Analysis of Hfq and SmpB Deletion Mutants in Salmonella: Implications for Virulence and Global Protein Translation

Using sample-matched transcriptomics and proteomics measurements it is now possible to begin to understand the impact of post-transcriptional regulatory programs in Enterobacteria. In bacteria post-transcriptional regulation is mediated by relatively few identified RNA-binding protein factors including CsrA, Hfq and SmpB. A mutation in any one of these three genes, csrA, hfq, and smpB, in Salmonella is attenuated for mouse virulence and unable to survive in macrophages. CsrA has a clearly defined specificity based on binding to a specific mRNA sequence to inhibit translation. However, the proteins regulated by Hfq and SmpB are not as clearly defined. Previous work identified proteins regulated by hfq using purification of the RNA-protein complex with direct sequencing of the bound RNAs and found binding to a surprisingly large number of transcripts. In this report we have used global proteomics to directly identify proteins regulated by Hfq or SmpB by comparing protein abundance in the parent and isogenic hfq or smpB mutant. From these same samples we also prepared RNA for microarray analysis to determine if alteration of protein expression was mediated post-transcriptionally. Samples were analyzed from bacteria grown under four different conditions; two laboratory conditions and two that are thought to mimic the intracellular environment. We show that mutants of hfq and smpB directly or indirectly modulate at least 20% and 4% of all possible Salmonella proteins, respectively, with limited correlation between transcription and protein expression. These proteins represent a broad spectrum of Salmonella proteins required for many biological processes including host cell invasion, motility, central metabolism, LPS biosynthesis, two-component regulatory systems, and fatty acid metabolism. Our results represent one of the first global analyses of post-transcriptional regulons in any organism and suggest that regulation at the translational level is widespread and plays an important role in virulence regulation and environmental adaptation for Salmonella

Mycobacterium tuberculosis ribosomal protein S1 (RpsA) and variants with truncated C-terminal end show absence of interaction with pyrazinoic acid.

Pyrazinamide (PZA) is an antibiotic used in first- and second-line tuberculosis treatment regimens. Approximately 50% of multidrug-resistant tuberculosis and over 90% of extensively drug-resistant tuberculosis strains are also PZA resistant. Despite the key role played by PZA, its mechanisms of action are not yet fully understood. It has been postulated that pyrazinoic acid (POA), the hydrolyzed product of PZA, could inhibit trans-translation by binding to Ribosomal protein S1 (RpsA) and competing with tmRNA, the natural cofactor of RpsA. Subsequent data, however, indicate that these early findings resulted from experimental artifact. Hence, in this study we assess the capacity of POA to compete with tmRNA for RpsA. We evaluated RpsA wild type (WT), RpsA ∆A438, and RpsA ∆A438 variants with truncations towards the carboxy terminal end. Interactions were measured using Nuclear Magnetic Resonance spectroscopy (NMR), Isothermal Titration Calorimetry (ITC), Microscale Thermophoresis (MST), and Electrophoretic Mobility Shift Assay (EMSA). We found no measurable binding between POA and RpsA (WT or variants). This suggests that RpsA may not be involved in the mechanism of action of PZA in Mycobacterium tuberculosis, as previously thought. Interactions observed between tmRNA and RpsA WT, RpsA ∆A438, and each of the truncated variants of RpsA ∆A438, are reported

Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P1′, glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates

The N-Terminus of GalE Induces tmRNA Activity in Escherichia coli

BACKGROUND: The tmRNA quality control system recognizes stalled translation complexes and facilitates ribosome recycling in a process termed 'ribosome rescue'. During ribosome rescue, nascent chains are tagged with the tmRNA-encoded SsrA peptide, which targets tagged proteins for degradation. In Escherichia coli, tmRNA rescues ribosomes arrested on truncated messages, as well as ribosomes that are paused during elongation and termination. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a new translational pausing determinant that leads to SsrA peptide tagging of the E. coli GalE protein (UDP-galactose 4-epimerase). GalE chains are tagged at more than 150 sites, primarily within distinct clusters throughout the C-terminal domain. These tagging sites do not correspond to rare codon clusters and synonymous recoding of the galE gene had little effect on tagging. Moreover, tagging was largely unaffected by perturbations that either stabilize or destabilize the galE transcript. Examination of GalE-thioredoxin (TrxA) fusion proteins showed that the GalE C-terminal domain is no longer tagged when fused to an N-terminal TrxA domain. Conversely, the N-terminus of GalE induced tagging within the fused C-terminal TrxA domain. CONCLUSIONS/SIGNIFICANCE: These findings suggest that translation of the GalE N-terminus induces subsequent tagging of the C-terminal domain. We propose that co-translational maturation of the GalE N-terminal domain influences ribosome pausing and subsequent tmRNA activity

Evidence That Two ATP-Dependent (Lon) Proteases in Borrelia burgdorferi Serve Different Functions

The canonical ATP-dependent protease Lon participates in an assortment of biological processes in bacteria, including the catalysis of damaged or senescent proteins and short-lived regulatory proteins. Borrelia spirochetes are unusual in that they code for two putative ATP-dependent Lon homologs, Lon-1 and Lon-2. Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted through the blood feeding of Ixodes ticks. Previous work in our laboratory reported that B. burgdorferi lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not. Because blood induction of Lon-1 may be of importance in the regulation of virulence factors critical for spirochete transmission, the clarification of functional roles for these two proteases in B. burgdorferi was the object of this study. On the chromosome, lon-2 is immediately downstream of ATP-dependent proteases clpP and clpX, an arrangement identical to that of lon of Escherichia coli. Phylogenetic analysis revealed that Lon-1 and Lon-2 cluster separately due to differences in the NH2-terminal substrate binding domains that may reflect differences in substrate specificity. Recombinant Lon-1 manifested properties of an ATP-dependent chaperone-protease in vitro but did not complement an E. coli Lon mutant, while Lon-2 corrected two characteristic Lon-mutant phenotypes. We conclude that B. burgdorferi Lons -1 and -2 have distinct functional roles. Lon-2 functions in a manner consistent with canonical Lon, engaged in cellular homeostasis. Lon-1, by virtue of its blood induction, and as a unique feature of the Borreliae, may be important in host adaptation from the arthropod to a warm-blooded host

The Lid Domain of Caenorhabditis elegans Hsc70 Influences ATP Turnover, Cofactor Binding and Protein Folding Activity

Hsc70 is a conserved ATP-dependent molecular chaperone, which utilizes the energy of ATP hydrolysis to alter the folding state of its client proteins. In contrast to the Hsc70 systems of bacteria, yeast and humans, the Hsc70 system of C. elegans (CeHsc70) has not been studied to date

Transfer–messenger RNA controls the translation of cell-cycle and stress proteins in Streptomyces

The transfer–messenger RNA (tmRNA)-mediated trans-translation mechanism is highly conserved in bacteria and functions primarily as a system for the rescue of stalled ribosomes and the removal of aberrantly produced proteins. Here, we show that in the antibiotic-producing soil bacterium Streptomyces coelicolor, trans-translation has a specialized role in stress management. Analysis of proteins that were carboxy-terminally His8-tagged by a recombinant tmRNA identified only 10 targets, including the stress proteins: DnaK heat-shock protein 70, thiostrepton-induced protein A, universal stress protein A, elongation factor Tu3, and the cell-cycle control proteins DasR, SsgA, SsgF and SsgR. Although tmRNA-tagged proteins are degraded swiftly, the translation of dnaK and dasR messenger RNAs (mRNAs) depends fully on tmRNA, whereas transcription is unaffected. The data unveil a surprisingly dedicated functionality for tmRNA, promoting the translation of the same mRNA it targets, at the expense of sacrificing the first nascent protein. In streptomycetes, tmRNA has evolved into a dedicated task force that ensures the instantaneous response to the exposure to stress

Crystal structure of the transfer-RNA domain of transfer-messenger RNA in complex with SmpB.

International audienceAccurate translation of genetic information into protein sequence depends on complete messenger RNA molecules. Truncated mRNAs cause synthesis of defective proteins, and arrest ribosomes at the end of their incomplete message. In bacteria, a hybrid RNA molecule that combines the functions of both transfer and messenger RNAs (called tmRNA) rescues stalled ribosomes, and targets aberrant, partially synthesized, proteins for proteolytic degradation. Here we report the 3.2-A-resolution structure of the tRNA-like domain of tmRNA (tmRNA(Delta)) in complex with small protein B (SmpB), a protein essential for biological functions of tmRNA. We find that the flexible RNA molecule adopts an open L-shaped conformation and SmpB binds to its elbow region, stabilizing the single-stranded D-loop in an extended conformation. The most striking feature of the structure of tmRNA(Delta) is a 90 degrees rotation of the TPsiC-arm around the helical axis. Owing to this unusual conformation, the SmpB-tmRNA(Delta) complex positioned into the A-site of the ribosome orients SmpB towards the small ribosomal subunit, and directs tmRNA towards the elongation-factor binding region of the ribosome. On the basis of this structure, we propose a model for the binding of tmRNA on the ribosome