Kinetic investigation of Escherichia coli RNA polymerase mutants that influence nucleotide discrimination and transcription fidelity

Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site, and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several Escherichia coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue betaAsp(675), which is >10A away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues betaArg(678) and betaAsp(814) do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the betaAsp(675) mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site.     

Study of highly constitutively active mutants suggests how cAMP activates cAMP receptor protein

The cAMP receptor protein (CRP) of Escherichia coli undergoes a conformational change in response to cAMP binding that allows it to bind specific DNA sequences. Using an in vivo screening method following the simultaneous randomization of the codons at positions 127 and 128 (two C-helix residues of the protein interacting with cAMP), we have isolated a series of novel constitutively active CRP variants. Sequence analysis showed that this group of variants commonly possesses leucine or methionine at position 127 with a beta-branched amino acid at position 128. One specific variant, T127L/S128I CRP, showed extremely high cAMP-independent DNA binding affinity comparable with that of cAMP-bound wild-type CRP. Further biochemical analysis of this variant and others revealed that Leu(127) and Ile(128) have different roles in stabilizing the active conformation of CRP in the absence of cAMP. Leu(127) contributes to an improved leucine zipper at the dimer interface, leading to an altered intersubunit interaction in the C-helix region. In contrast, Ile(128) stabilizes the proper position of the beta4/beta5 loop by functionally communicating with Leu(61). By analogy, the results suggest two direct local effects of cAMP binding in the course of activating wild-type CRP: (i) C-helix repositioning through direct interaction with Thr(127) and Ser(128) and (ii) the concomitant reorientation of the beta4/beta5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.     

Spatial and temporal expression of the response regulators ARR22 and ARR24 in Arabidopsis thaliana

ARR22 (At3g04280) is a novel Type A response regulator whose function in Arabidopsis is unknown. RT-PCR analysis has shown that expression of the gene takes place in flowers and developing pods with the tissues accumulating different proportions of splice variants. Spatial analysis of expression, using ARR22::GUS plants as a marker, has revealed that the reporter protein accumulates specifically at the junction between the funiculus and the chalazal tissue. Expression can be up-regulated at this location by wounding the developing seed. A detailed analysis has failed to detect ARR22 expression at any other sites and, to support this assertion, the only evidence for tissue ablation in ARR22::Barnase plants is during seed development, with the consequence that embryo growth is attenuated. Ectopic expression of ARR22, driven by either the CaMV 35S or the pea plastocyanin (PPC) promoters, resulted in the generation of plants exhibiting extremely stunted root and shoot growth. No viable progeny could be isolated from the PPC::ARR22 transgenic lines. An RT-PCR analysis of a recently annotated gene (ARR24-At5g26594), that exhibits 66% amino acid similarity to ARR22, has shown that expression is also predominantly in floral and silique tissues. Examination of ARR24::GUS plants has revealed that the activity of the promoter is primarily restricted to pollen grains indicating that this gene is unlikely to display an overlapping function with ARR22. Analyses of individual KO lines of either ARR22 or ARR24 have failed to identify a mutant phenotype under the growth conditions employed and the double knockout ARR22/ARR24 line is also indistinguishable from wild-type plants. These results are discussed in the light of the proposed role of response regulators in plant growth and development.     

Memory T cell populations in the lung airways are maintained by continual recruitment

Effector memory T cell populations in the periphery play a key role in cellular immune responses to secondary infections. However, it is unclear how these populations are maintained under steady-state conditions in nonlymphoid peripheral sites, such as the lung airways. In this study, we show that LFA-1 expression is selectively down-regulated following entry of memory T cells into the lung airways. Using Sendai virus as a mouse model of respiratory virus infection, we use LFA-1 expression levels to demonstrate that effector memory T cell populations in the lung airways are maintained by continual recruitment of new cells from the circulation. The rate of memory cell recruitment is surprisingly rapid, resulting in replacement of 90% of the population every 10 days, and is maintained for well over 1 year following viral clearance. These data indicate that peripheral T cell memory is dynamic and depends on a systemic source of T cells.     

Cell-free immune reactions in insects

Insects, like many other multicellular organisms, are able to recognise and inactivate potential pathogens and toxins in the absence of cells. Here we show that the recognition and inactivation of lipopolysaccharides (LPS) and bacteria is mediated by lipophorin particles, which are the lipid carrier in insects. In immune-induced insects sub-populations of lipophorin particles are associated with pattern recognition proteins and regulatory proteins that activate prophenoloxidase. Moreover, interactions with lectins result in the assembly of lipophorin particles into cage-like coagulation products, effectively protecting the surrounding tissues and cells from the potentially damaging effects of pathogens and phenoloxidase products. The existence of cell-free defence reactions implies that immune signals exist upstream of cell-bound receptors.     

The poor growth of Rhodospirillum rubrum mutants lacking PII proteins is due to an excess of glutamine synthetase activity

The P(II) family of proteins is found in all three domains of life and serves as a central regulator of the function of proteins involved in nitrogen metabolism, reflecting the nitrogen and carbon balance in the cell. The genetic elimination of the genes encoding these proteins typically leads to severe growth problems, but the basis of this effect has been unknown except with Escherichia coli. We have analysed a number of the suppressor mutations that correct such growth problems in Rhodospirillum rubrum mutants lacking P(II) proteins. These suppressors map to nifR3, ntrB, ntrC, amtB(1) and the glnA region and all have the common property of decreasing total activity of glutamine synthetase (GS). We also show that GS activity is very high in the poorly growing parental strains lacking P(II) proteins. Consistent with this, overexpression of GS in glnE mutants (lacking adenylyltransferase activity) also causes poor growth. All of these results strongly imply that elevated GS activity is the causative basis for the poor growth seen in R. rubrum mutants lacking P(II) and presumably in mutants of some other organisms with similar genotypes. The result underscores the importance of proper regulation of GS activity for cell growth.     

Seduced by the dark side: integrating molecular and ecological perspectives on the influence of light on plant defence against pests and pathogens

Plants frequently suffer attack from herbivores and microbial pathogens, and have evolved a complex array of defence mechanisms to resist defoliation and disease. These include both preformed defences, ranging from structural features to stores of toxic secondary metabolites, and inducible defences, which are activated only after an attack is detected. It is well known that plant defences against pests and pathogens are commonly affected by environmental conditions, but the mechanisms by which responses to the biotic and abiotic environments interact are only poorly understood. In this review, we consider the impact of light on plant defence, in terms of both plant life histories and rapid scale molecular responses to biotic attack. We bring together evidence that illustrates that light not only modulates defence responses via its influence on biochemistry and plant development but, in some cases, is essential for the development of resistance. We suggest that the interaction between the light environment and plant defence is multifaceted, and extends across different temporal and biological scales.     

Cell-cell adhesion in fresh sugar-beet root parenchyma requires both pectin esters and calcium cross-links

Multicellular plants depend for their integrity on effective adhesion between their component cells. This adhesion depends upon various cross-links; ionic, covalent or weak interactions between the macromolecules of the adjacent cell walls. In sugar-beet ( Beta vulgaris L. Aztec) root parenchyma, cell-cell adhesion is disrupted by successive extractions with a calcium-chelating agent (imidazole) and a de-esterifying agent (sodium carbonate) but not by the calcium-chelating agent or the de-esterifying agent alone. Cell-cell adhesion in sugar-beet parenchyma thus depends upon both ester and Ca²⁺ cross-linked polymers. Pectic polysaccharides are removed by these treatments. Both parallel-electron energy-loss spectroscopy (PEELS) and Image-EELS show that calcium-binding sites are removed from the wall by imidazole. Using a monoclonal antibody that recognizes a relatively unesterified epitope of homogalacturonan, JIM 5, we show that a subset of JIM 5-reactive antigens remain in the middle lamella after Ca²⁺ chelation and that this subset is removed by cold (4° C) Na₂CO₃-induced breakage of ester bonds. Fourier transform infrared, nuclear magnetic resonance, and spectrophotometric assays show that methyl and feruloyl esters are removed from the wall by Na₂CO₃ but acetyl esters remain. Sodium carbonate extraction at 20° C removes cell wall autofluorescence and most of the feruloylated moieties from the wall. We propose that the chelator-resistant subset of ester-linked JIM 5-reactive pectins are important for cell-cell adhesion.     

A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration

Zebrafish possess a unique yet poorly understood capacity for cardiac regeneration. Here, we show that regeneration proceeds through two coordinated stages following resection of the ventricular apex. First a blastema is formed, comprised of progenitor cells that express precardiac markers, undergo differentiation, and proliferate. Second, epicardial tissue surrounding both cardiac chambers induces developmental markers and rapidly expands, creating a new epithelial cover for the exposed myocardium. A subpopulation of these epicardial cells undergoes epithelial-to-mesenchymal transition (EMT), invades the wound, and provides new vasculature to regenerating muscle. During regeneration, the ligand fgf17b is induced in myocardium, while receptors fgfr2 and fgfr4 are induced in adjacent epicardial-derived cells. When fibroblast growth factors (Fgf) signaling is experimentally blocked by expression of a dominant-negative Fgf receptor, epicardial EMT and coronary neovascularization fail, prematurely arresting regeneration. Our findings reveal injury responses by myocardial and epicardial tissues that collaborate in an Fgf-dependent manner to achieve cardiac regeneration.