Andrea Parmeggiani

Phase coexistence in driven one-dimensional transport

We study a one-dimensional totally asymmetric exclusion process with random particle attachments and detachments in the bulk. The resulting dynamics leads to unexpected stationary regimes for large but finite systems. Such regimes are characterized by a phase coexistence of low and high density regions separated by domain walls. We use a mean-field approach to interpret the numerical results obtained by Monte Carlo simulations, and we predict the phase diagram of this nonconserved dynamics in the thermodynamic limit.

Properties and regulation of the GTPase activities of elongation factors Tu and G, and of initiation factor 2

SummaryDuring protein synthesis the interaction with ribosomes of elongation factors Tu (EF-Tu), G (EF-G) and initiation factor 2 (IF-2) is associated with the hydrolysis of GTP which is directly related to the functions of the factors. In this article we review systematically the properties of these GTPase activities in the presence and absence of protein synthesis, and by examining the characteristics of the different minimal systems for the expression of these activities we point to the role of the various effectors and to the enzymological aspects of the systems. For EF-Tu, it has been possible to eliminate any requirement for macromolecular effectors, showing that the factor itself is a GTPase. For EF-G, the presence of at least the 50S ribosomal subunit has remained a requirement, whereas IF-2 needs both the 50S and 30S subunits to exibit GTPase activity. Between the GTPase activities of the three factors there are some striking similarities, but important differences prevail as a consequence of the specificity of the different functions. This can also be seen by examining the respective ribosomal regions implicated in these reactions. When coupled with protein synthesis, the three GTPase activities reveal characteristics differing from those observed in partial systems.

Relevance of histidine-84 in the elongation factor Tu GTPase activity and in poly(Phe) synthesis: Its substitution by glutamine and alanine

Substitution of His‐84 (→ Gln and → Ala), a residue of the switch II region of E. coli elongation factor (EF) Tu, hardly affected the binding of GTP or GDP. The activity in poly(Phe) synthesis and GTP hydrolysis of EF‐Tu H84Q were both reduced to about 35%, as compared to EF‐Tu wt, whereas EF‐Tu H84A was inactive in poly(Phe) synthesis but still showed a 10% residual GTPase activity. Phe‐tRNAPhe exerted a similar inhibitory effect on the GTPase activity of EF‐Tu wt and EF‐Tu H84Q while abolishing that of EF‐Tu H84A. Ribosomes enhanced the GTPase activity of EF‐Tu H84Q, but not that of EF‐Tu H84A, on which they even seemed to exert an inhibitory effect. The one‐round GTP hydrolysis associated with the EF‐TuH84Q‐dependent binding of Phe‐tRNAPhe to poly(U)‐programmed ribosomes was less efficient than with EF‐Tu wt. Kirromycin stimulated the GTPase activities of both mutants less than EF‐Tu wt. The results of this work do not support a catalytic role of His‐84 in the intrinsic GTPase of EF‐Tu, but they emphasize the importance of its side‐chain for polypeptide synthesis and GTP hydrolysis.

Enacyloxin IIa pinpoints a binding pocket of elongation factor Tu for development of novel antibiotics

Elongation factor (EF-) Tu·GTP is the carrier of aminoacyl-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu·GDP from the ribosome. The crystal structure of the Escherichia coli EF-Tu·guanylyl iminodiphosphate (GDPNP)·enacyloxin IIa complex at 2.3 Å resolution presented here reveals the location of the antibiotic at the interface of domains 1 and 3. The binding site overlaps that of kirromycin, an antibiotic with a structure that is unrelated to enacyloxin IIa but that also inhibits EF-Tu·GDP release. As one of the major differences, the enacyloxin IIa tail borders a hydrophobic pocket that is occupied by the longer tail of kirromycin, explaining the higher binding affinity of the latter. EF-Tu·GDPNP·enacyloxin IIa shows a disordered effector region that in the Phe-tRNAPhe·EF-Tu (Thermus aquaticus)·GDPNP·enacyloxin IIa complex, solved at 3.1 Å resolution, is stabilized by the interaction with tRNA. This work clarifies the structural background of the action of enacyloxin IIa and compares its properties with those of kirromycin, opening new perspectives for structure-guided design of novel antibiotics.

Mechanisms of EF-Tu, a pioneer GTPase.

This review considers several aspects of the function of EF-Tu, a protein that has greatly contributed to the advancement of our knowledge of both protein biosynthesis and GTP-binding proteins in general. A number of topics are described with emphasis on the function-structure relationships, in particular of EF-Tus domains, the nucleotide-binding site, and the magnesium-binding network. Aspects related to the interaction with macromolecular ligands and antibiotics and to folding and GTPase activity are also presented and discussed. Comments and criticism are offered to draw attention to remaining discrepancies and problems.

Totally asymmetric simple exclusion process on networks.

We study the totally asymmetric simple exclusion process (TASEP) on complex networks, as a paradigmatic model for transport subject to excluded volume interactions. Building on TASEP phenomenology on a single segment and borrowing ideas from random networks we investigate the effect of connectivity on transport. In particular, we argue that the presence of disorder in the topology of vertices crucially modifies the transport features of a network: irregular networks involve homogeneous segments and have a bimodal distribution of edge densities, whereas regular networks are dominated by shocks leading to a unimodal density distribution. The proposed numerical approach of solving for mean-field transport on networks provides a general framework for studying TASEP on large networks, and is expected to generalize to other transport processes.

Variable Combinations of Specific Ephrin Ligand/Eph Receptor Pairs Control Embryonic Tissue Separation

Vertebrate embryonic cells recognize self from non-self, thus restricting repulsion at tissue boundaries, through a combination of multiple ephrins and Eph receptors, simply based on binding selectivity and asymmetric expression.

Stochastic Self-Assembly of ParB Proteins Builds the Bacterial DNA Segregation Apparatus

Many canonical processes in molecular biology rely on the dynamic assembly of higher-order nucleoprotein complexes. In bacteria, the assembly mechanism of ParABS, the nucleoprotein super-complex that actively segregates the bacterial chromosome and many plasmids, remains elusive. We combined super-resolution microscopy, quantitative genome-wide surveys, biochemistry, and mathematical modeling to investigate the assembly of ParB at the centromere-like sequences parS. We found that nearly all ParB molecules are actively confined around parS by a network of synergistic protein-protein and protein-DNA interactions. Interrogation of the empirically determined, high-resolution ParB genomic distribution with modeling suggests that instead of binding only to specific sequences and subsequently spreading, ParB binds stochastically around parS over long distances. We propose a new model for the formation of the ParABS partition complex based on nucleation and caging: ParB forms a dynamic lattice with the DNA around parS. This assembly model and approach to characterizing large-scale, dynamic interactions between macromolecules may be generalizable to many unrelated machineries that self-assemble in superstructures.

Modeling cytoskeletal traffic: an interplay between passive diffusion and active transport.

We introduce the totally asymmetric simple exclusion process with Langmuir kinetics on a network as a microscopic model for active motor protein transport on the cytoskeleton, immersed in the diffusive cytoplasm. We discuss how the interplay between active transport along a network and infinite diffusion in a bulk reservoir leads to a heterogeneous matter distribution on various scales: we find three regimes for steady state transport, corresponding to the scale of the network, of individual segments, or local to sites. At low exchange rates strong density heterogeneities develop between different segments in the network. In this regime one has to consider the topological complexity of the whole network to describe transport. In contrast, at moderate exchange rates the transport through the network decouples, and the physics is determined by single segments and the local topology. At last, for very high exchange rates the homogeneous Langmuir process dominates the stationary state. We introduce effective rate diagrams for the network to identify these different regimes. Based on this method we develop an intuitive but generic picture of how the stationary state of excluded volume processes on complex networks can be understood in terms of the single-segment phase diagram.

The GTPase activity of elongation factor Tu and the 3'‐terminal end of aminoacyl‐tRNA

In the elongation cycle, the binding of the ternary complex EF-Tu . GTP . aa-tRNA to the mRNA . ribosome complex is accompanied by the hydrolysis of GTP, a prerequisite for the release of EF-Tu from the ribosome (review [ 1 I). We have utilized the antibiotic kirromycin to analyze the role of the individual components of the EF-Tudependent GTPase reaction, since this compound allows EF-Tu to support a turnover of GTPase activity which is specifically stimulated by aa-tRNA and ribosomes [2-61. As in the physiological system, only aminoacylated tRNA and not deacylated tRNA orN-acetylaminoacyl-tRNA displays an effect in the kirromycin-dependent activity. Kirromycin can be therefore a useful tool to study the involvement of the 3’-terminal end of aa-tRNA in the GTPase activity of EF-Tu. Here, we show that 3’-terminal aa-tRNA fragments, containing l-5 residues, can stimulate the GTPase activity of EF-Tu induced by kirromycin. The effect, which occurs already with 2’(3’)-O-L-aminoacyladenosine, is increased by the presence of the other residues, the penultimate one playing a critical role. Ribosomes enhance the sensitivity of the system.