Rebecca Keller

Poly(A) Tail Length-dependent Stabilization of GAP-43 mRNA by the RNA-binding Protein HuD

The neuronal ELAV-like RNA-binding protein HuD binds to a regulatory element in the 3′-untranslated region of the growth-associated protein-43 (GAP-43) mRNA. Here we report that overexpression of HuD protein in PC12 cells stabilizes the GAP-43 mRNA by delaying the onset of mRNA degradation and that this process depends on the size of the poly(A) tail. Using a polysome-basedin vitro mRNA decay assay, we found that addition of recombinant HuD protein to the system increased the half-life of full-length, capped, and polyadenylated GAP-43 mRNA and that this effect was caused in part by a decrease in the rate of deadenylation of the mRNA. This stabilization was specific for GAP-43 mRNA containing the HuD binding element in the 3′-untranslated region and a poly(A) tail of at least 150 A nucleotides. In correlation with the effect of HuD on GAP-43 mRNA stability, we found that HuD binds GAP-43 mRNAs with long tails (A150) with 10-fold higher affinity than to those with short tails (A30). We conclude that HuD stabilizes the GAP-43 mRNA through a mechanism that is dependent on the length of the poly(A) tail and involves changes in its affinity for the mRNA.

Signal integration by the Cpx-envelope stress system

The Cpx-envelope stress system coordinates the expression and assembly of surface structures important for the virulence of Gram-negative pathogenic bacteria. It is comprised of the membrane-anchored sensor kinase CpxA, the cytosolic response regulator CpxR and the accessory protein CpxP. Characteristic of the group of two-component systems, the Cpx system responds to a broad range of stimuli including pH, salt, metals, lipids and misfolded proteins that cause perturbation in the envelope. Moreover, the Cpx system has been linked to inter-kingdom signalling and bacterial cell death. However, although signal specificity has been assumed, for most signals the mechanism of signal integration is not understood. Recent structural and functional studies provide the first insights into how CpxP inhibits CpxA and serves as sensor for misfolded pilus subunits, pH and salt. Here, we summarize and reflect on the current knowledge on signal integration by the Cpx-envelope stress system.

Structural Basis for Two-component System Inhibition and Pilus Sensing by the Auxiliary CpxP Protein.

Bacteria are equipped with two-component systems to cope with environmental changes, and auxiliary proteins provide response to additional stimuli. The Cpx two-component system is the global modulator of cell envelope stress in Gram-negative bacteria that integrates very different signals and consists of the kinase CpxA, the regulator CpxR, and the dual function auxiliary protein CpxP. CpxP both inhibits activation of CpxA and is indispensable for the quality control system of P pili that are crucial for uropathogenic Escherichia coli during kidney colonization. How these two essential biological functions of CpxP are linked is not known. Here, we report the crystal structure of CpxP at 1.45 Å resolution with two monomers being interdigitated like “left hands” forming a cap-shaped dimer. Our combined structural and functional studies suggest that CpxP inhibits the kinase CpxA through direct interaction between its concave polar surface and the negatively charged sensor domain on CpxA. Moreover, an extended hydrophobic cleft on the convex surface suggests a potent substrate recognition site for misfolded pilus subunits. Altogether, the structural details of CpxP provide a first insight how a periplasmic two-component system inhibitor blocks its cognate kinase and is released from it.

Molecular dissection of TatC defines critical regions essential for protein transport and a TatB–TatC contact site

The twin arginine transport (Tat) system transports folded proteins across the prokaryotic cytoplasmic membrane and the plant thylakoid membrane. TatC is the largest and most conserved component of the Tat machinery. It forms a multisubunit complex with TatB and binds the signal peptides of Tat substrates. Here we have taken a random mutagenesis approach to identify substitutions in Escherichia coli TatC that inactivate protein transport. We identify 32 individual amino acid substitutions that abolish or severely compromise TatC activity. The majority of the inactivating substitutions fall within the first two periplasmic loops of TatC. These regions are predicted to have conserved secondary structure and results of extensive amino acid insertion and deletion mutagenesis are consistent with these conserved elements being essential for TatC function. Three inactivating substitutions were identified in the fifth transmembrane helix of TatC. The inactive M205R variant could be suppressed by mutations affecting amino acids in the transmembrane helix of TatB. A physical interaction between TatC helix 5 and the TatB transmembrane helix was confirmed by the formation of a site‐specific disulphide bond between TatC M205C and TatB L9C variants. This is the first molecular contact site mapped to single amino acid level between these two proteins.

Co-operation between different targeting pathways during integration of a membrane protein

The Sec and Tat pathways are both required to insert the three hydrophobic domains of the Rieske protein into the membrane.

Scanning tunneling microscopy images of metal‐coated bacteriophages and uncoated, double‐stranded DNA

One of the primary goals of scanning tunneling microscopy (STM) work in biology is to obtain high‐resolution images of biological molecules under conditions that resemble those in vivo. To accomplish this goal two main difficulties need to be overcome. (1) Problems caused by the low conductivity of biological material, and (2) depositing the sample in a controlled and reliable way on a flat, inert conducting substrate. The first problem can be solved by coating the sample with a thin metal layer, at some cost in resolution. This allows the second problem, controllable depositing the sample, to be approached systematically, independent of difficulties caused by low conductivity. Using this approach, two different kinds of viruses have been imaged, bacteriophage T7 and fd. STM images of intact T7 and fd bacteriophages, and lysed T7 phages have been obtained in a straightforward and routine way. To obtain high‐resolution images it is necessary to use uncoated samples. The conductivity problems cannot be avoi...

A method for imaging E. coli. RNA polymerase holoenzyme with the scanning tunneling microscope in an aqueous environment

An electodeposition method has been developed for depositing E. coli. RNA polymerase onto a monoatomically flat gold surface. The scanning tunneling microscope has been used to image this enzyme in a high‐humidity or water/glycerol environment, which allows direct observation without metal coating. The deposition technique is reproducible enough to allow optimization of the deposition parameters for controlled electrodeposition. Time‐dependence studies were conducted to help determine the binding mechanism of the protein. The enzyme molecules appear as ordered arrays, amorphous features, or ‘‘jaw‐shaped’’ molecules depending on the deposition conditions used. The ‘‘jaw‐shaped’’ molecules closely resemble those observed by electron microscopy. In some of these a groove 22 A wide can be seen.

The Escherichia coli Envelope Stress Sensor CpxA Responds to Changes in Lipid Bilayer Properties

The Cpx stress response system is induced by various environmental and cellular stimuli. It is also activated in Escherichia coli strains lacking the major phospholipid, phosphatidylethanolamine (PE). However, it is not known whether CpxA directly senses changes in the lipid bilayer or the presence of misfolded proteins due to the lack of PE in their membranes. To address this question, we used an in vitro reconstitution system and vesicles with different lipid compositions to track modulations in the activity of CpxA in different lipid bilayers. Moreover, the Cpx response was validated in vivo by monitoring expression of a PcpxP-gfp reporter in lipid-engineered strains of E. coli. Our combined data indicate that CpxA responds specifically to different lipid compositions.

Electrodeposition procedure of E. coli RNA polymerase onto gold and deposition of E. coli RNA polymerase onto mica for observation with scanning force microscopy

Molecules of the transcriptional enzyme E. coli RNA polymerase (RNAP) have been deposited using three different deposition methods: (1) passive adsorption onto gold, (2) electrochemical adsorption onto gold and (3) adsorption onto mica. In all cases SFM imaging was straightforward and reliable, and surface coverage by the protein varied with deposition conditions as expected. To determine the nature of the electrochemical treatment on the gold substrate, cyclic voltammetry was performed with various chemical solutions. Finally, a comparison is made between the SFM images of RNAP obtained with these methods and STM images obtained earlier. Both STM and SFM show strikingly similar results; however, heights and widths of individual molecules differ.

Post-Transcriptional Control of the GAP-43 mRNA by the ELAV-Like Protein HuD

Post-transcriptional control by mRNA-binding proteins is critical for shaping the temporal and spatial pattern of expression of a large number of developmentally regulated genes. Among these is the gene for GAP-43, a growth-associated protein expressed in neurons primarily during the initial establishment and remodeling of neural connections. Both transcriptional and post-transcriptional mechanisms control GAP-43 gene expression during development. While promoter activity determines the neural-specific expression of the gene, changes in mRNA stability modulate GAP-43 expression in neurons undergoing process outgrowth in response to growth factors and other signaling agents. For example, in PC12 cells induced to differentiate by nerve growth factor (NGF), GAP-43 mRNA levels are regulated primarily through selective changes in the rate of degradation of the mRNA. This process depends on the activation of protein kinase C (PKC) and is mediated by the interaction of highly conserved sequences in the 3’ untranslated region (3 ‘UTR) of the mRNA with neuronal-specific RNA-binding proteins. One of these proteins was recently identified as the ELAV-like protein HuD. SLAV is an RNA-binding protein that is critical for the development of the nervous system in Drosophila and HuD is one of four human homologs of this protein. This chapter discusses the evidence demonstrating a role for HuD in the control of GAP-43 mRNA stability, gene expression and neuronal differentiation, and presents recent findings on the molecular mechanisms underlying these effects.