The structure of bacteriophage T4-specific messenger RNAs. I. Tightly folded conformation revealed by sedimentation in denaturing solvents.

Measurements of the sedimentation velocity under completely denaturing conditions in dimethylsulfoxide were used to obtain the molecular weights of some T4-specific mRNAs including the biologically active lysozyme mRNA. The amount of folding was determined by comparing their sedimentation in sucrose gradients in the presence and absence of this reagent. T4-specific mRNAs sediment much more slowly in the presence of dimethylsulfoxide than in its absence. This difference is linearly dependent on molecular weight; the dependence was similar to that observed for Escherichia coli stable RNA controls and very different from that of random coils (E. coli stable RNA treated with formaldehyde so that no base-pairing is possible). We conclude that the T4-specific mRNAs are not random coils but resemble much more closely the highly base-paired E. coli stable RNAs.     

Evolution of regulatory enzymes towards functional simplicity

The potential kinetic complexity of polymeric regulatory enzymes does not seem to be often expressed in nature. Most of these enzymes exhibit in fact a rather simple kinetic behaviour. This functional simplicity is probably the consequence of constraints between rate constants or of blocking of some reaction steps. Functional simplicity is believed to have emerged in the course of neo-Darwinian evolution as a consequence of a trend towards an improved functional efficiency. Functional efficiency may be reached, in polymeric regulatory enzymes, when either of the two sets of conditions are met. The first set of conditions implies the occurrence of the unicity of enzyme conformation in any transition state, a loose coupling between subunits and an exact balance of the driving forces exerted by the enzyme in the forward and backward directions of the catalytic step. This situation results in constraints between rate constants which allow degenerescence of the steady state rate equation. The second set of conditions involves again the unicity of enzyme conformation in any of the transition states, associated with a tight coupling of subunits, and a driving force exerted by the enzyme much strongly in the forward than in the backward direction of the catalytic step. These conditions imply blocking of some reaction steps and again degenerescence of the corresponding rate equation. The most frequent types of quaternary structure and subunit interactions, namely loose coupling between subunits, and tight coupling associated with conservation of at least one symmetry axis, have probably emerged as molecular organizations, which precisely allow both functional efficiency and simplicity to occur. Indeed these situations probably represent the term of two different evolutionary trends. Therefore enzymes that have not reached this state usually exhibit more complex kinetic behaviour. Wavy curves, “bumps” and turning points may be considered as manifestations of the ancestral character of an enzyme.     

Kinetics of the conformational transition of the spinach chloroplast fructose-1,6-bisphosphatase induced by fructose 2,6-bisphosphate.

The activation of oxidized chloroplast fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate and magnesium previously described at pH 7.5 [Soulié et al. (1988) Eur. J. Biochem. 176, 111-117] has now been studied at pH 8, the pH which prevails under light conditions in the chloroplast stroma. The process obeys a hysteretic mechanism but the rate of activation is considerably increased with half-times down to 50 s and the apparent dissociation constant of fructose 2,6-bisphosphate from the enzyme is lowered from 1 mM at pH 7.5 to 3.3 microM at pH 8. The process is strictly metal-dependent with a half-saturation concentration of 2.54 mM for magnesium. The conformational transition postulated in our hysteretic model has been investigated through both the spectrophometric and chemical modification approaches. The activation of the enzyme by fructose 2,6-bisphosphate in the presence of magnesium results in a slow modification of the ultraviolet absorption spectrum of the enzyme with an overall increase of 3% at 290 nm. The same treatment leads to the protection of two free sulfhydryls and an increased reactivity of one sulfhydryl group/enzyme monomer to modification by 5,5'-dithiobis(2-nitrobenzoic acid). The titration of the exposed cysteinyl residue prevents the relaxation of enzyme species induced by fructose 2,6-bisphosphate to the native form. The activation of chloroplast fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate is discussed both with respect to the understanding of the overall regulation properties of the enzyme and to a possible physiological significance of this process.     

Increased hexose transport in the roots of tomato plants submitted to prolonged hypoxia

We investigated the effects of prolonged hypoxia on the sugar uptake in tomato (Solanum lycopersicum L. var. MP-1) roots. Hydroponic cultures of whole tomato plants were submitted to hypoxic treatment for 1 week, and the roots were analyzed for sugar concentrations, hexose uptake and hexose transporter expression level. Contrary to what has been observed after anoxic shock or short-term hypoxic treatment, we show that sugar concentrations increase and hexose uptake is up-regulated in the roots after 1 week of hypoxic treatment. Increased hexose transport is concomitant with the induction of the hexose transporter gene LeHT2. These responses may be due either to a direct effect of low O₂ supply, or to a secondary effect associated with the increase in sugar concentrations, which, typically, develops in most hypoxic plants.     

Why are cellular switches Boolean? General conditions for multistable genetic circuits

The logic of cellular decision-making is largely controlled by regulatory circuits defining molecular switches. Such switching elements allow to turn a graded input signal into an all-or-nothing output. Traditional studies have focused on this bistable picture of regulation, but higher-order scenarios involving tristable and tetrastable states are possible too. Are these multiswitches allowed in simple gene regulatory networks? Are they likely to be observed? If not, why not? In this paper we present the examination of this question by means of a simple but powerful geometric approach. We examine the relation between multistability, the degree of multimerization of the regulators and the role of autoloops within a deterministic setting, finding that N-stable circuits are possible, although their likelihood to occur rapidly decays with the order of the switch. Our work indicates that, despite two-component circuits are able to implement multistability, they are optimal for Boolean switches. The evolutionary implications are outlined.     

Information catastrophe in RNA viruses through replication thresholds

RNA viruses are known to replicate at very high mutation rates. These rates are actually known to be close to their so-called error threshold. This threshold is in fact a critical point beyond which genetic information is lost through a so-called error catastrophe. However, the transition from a stable quasispecies to genetic drift and loss of information can also occur by crossing replication thresholds, below some replication rates, the viral population is suddenly unable to survive. Available data from hepatitis C virus population analysis [Mas, A., Ulloa, E., Bruguera, M., Furci?, I., Garriga, D., Fábregas, S., Andreu, D., Saiz, J.C., Díez, J., 2004. Hepatitis C virus population analysis of a single-source nosocomial outbreak reveals an inverse correlation between viral load and quasispecies complexity. J. Gen. Virol. 85, 3619-3626] can be interpreted through this theoretical view, providing evidence for such a replication threshold. Here a simple model is used in order to provide evidence for such a phenomenon, consistent with available data.