Translational coupling at an intercistronic boundary of the Escherichia coli galactose operon

Precise frameshift and nonsense mutations were introduced into the region preceding the galactokinase gene (galK) of Escherichia coli. These mutations after the position at which upstream translation terminates relative to the galK translation initiation signal. Constructions were characterized that allow ribosomes to stop selectively before, within or downstream from the galK initiation signal. The effects of these mutations on galK expression were monitored. Galactokinase levels are highest when upstream translation terminates within the galK initiation region. In contrast, when translation stops either upstream or down stream from the galK start site, galK expression is drastically reduced. These results demonstrate that the galK gene is translationally coupled to the gene immediately preceding galK in the gal operon (that is, galT), and that the coupling effect depends primarily on the position at which upstream translation terminates relative to the galK start site. Possible mechanisms and implications of this translational coupling phenomenon are discussed.     

Finding the optimal lengths for three branches at a junction

This paper presents an exact analytical solution to the problem of locating the junction point between three branches so that the sum of the total costs of the branches is minimized. When the cost per unit length of each branch is known the angles between each pair of branches can be deduced following reasoning first introduced to biology by Murray. Assuming the outer ends of each branch are fixed, the location of the junction and the length of each branch are then deduced using plane geometry and trigonometry. The model has applications in determining the optimal cost of a branch or branches at a junction. Comparing the optimal to the actual cost of a junction is a new way to compare cost models for goodness of fit to actual junction geometry. It is an unambiguous measure and is superior to comparing observed and optimal angles between each daughter and the parent branch. We present data for 199 junctions in the pulmonary arteries of two human lungs. For the branches at each junction we calculated the best fitting value of x from the relationship that flow ∞ (radius)^x. We found that the value of x determined whether a junction was best fitted by a surface, volume, drag or power minimization model. While economy of explanation casts doubt that four models operate simultaneously, we found that optimality may still operate, since the angle to the major daughter is less than the angle to the minor daughter. Perhaps optimality combined with a space filling branching pattern governs the branching geometry of the pulmonary artery.     

Non-canonical RNA arrangement in T4-even phages: accommodated ribosome binding site at the gene 26-25 intercistronic junction

Translational initiation region of bacteriophage T4 gene 25 contains three potential Shine and Dalgarno sequences: SD1, SD2 and SD3. Mutational analysis has predicted that an mRNA stem-loop structure may include SD1 and SD2, bringing the most typical sequence SD3, GAGG, to the initiation codon. Here, we report physical evidence demonstrating that previously predicted mRNA stem-loop structure indeed exists in vivo during gene 25 expression in T4-infected Escherichia coli cells. The second mRNA stem-loop structure is identified 14 nucleotides upstream of the stem-loop I, while the SD3 sequence, as well as the start codon of the gene, are proved to be within an unfolded stretch of mRNA. Phylogenetic comparison of 38 T4-like phages reveals that the T-even and some pseudoT-even phages evolve a similar structural strategy for the translation initiation of 25 , while pseudoT-even, schizoT-even and exoT-even phages use an alternative mRNA arrangement. Taken together, the results indicate that a specific mRNA fold forms the split ribosome binding site at the gene 26-25 intercistronic junction, which is highly competent in the translational initiation. We conclude that this ribosome binding site has evolved after T-even diverged from other T4-like phages. Additionally, we determine that the SD sequence GAGG is most widespread in T4.     

Streptomycin interferes with conformational coupling between codon recognition and GTPase activation on the ribosome

Aminoacyl-tRNAs (aa-tRNAs) are selected by the ribosome through a kinetically controlled induced fit mechanism. Cognate codon recognition induces a conformational change in the decoding center and a domain closure of the 30S subunit. We studied how these global structural rearrangements are related to tRNA discrimination by using streptomycin to restrict the conformational flexibility of the 30S subunit. The antibiotic stabilized aa-tRNA on the ribosome both with a cognate and with a near-cognate codon in the A site. Streptomycin altered the rates of GTP hydrolysis by elongation factor Tu (EF-Tu) on cognate and near-cognate codons, resulting in almost identical rates of GTP hydrolysis and virtually complete loss of selectivity. These results indicate that movements within the 30S subunit at the streptomycin-binding site are essential for the coupling between base pair recognition and GTP hydrolysis, thus modulating the fidelity of aa-tRNA selection.     

Gap junction blockage interferes with neuronal and astroglial differentiation of mouse P19 embryonal carcinoma cells

During embryonic development, cells not only increase in number, they also undergo specialization and differentiate into diverse cell types that are organized into different tissues and organs. Nervous system development, for example, involves a complex series of events such as neuronal and astroglial differentiation that are coordinated among adjacent cells. The organization of growth and differentiation may be mediated, at least partly, by exchange of small ions and molecules via intercellular gap junction channels. These structures are mode of connexons (hemichannels), which are hexameric assemblies of the gap junction proteins, connexins. We investigated the role of intercellular communication in neuronal and astroglial differentiation by using a gap junction blocking agent, carbenoxolone (CBX), in comparison to its inactive (control) analog, glycyrrhizic acid (GZA). We used the mouse P19 embryonal carcinoma cell line, which differentiates into neurons and astrocytes upon retinoic acid (RA) induction. Our results show that both GZA- and CBX-treated cells express alpha 1 connexin (connexin43). The level of alpha 1 connexin decreases upon RA induction. CBX treated cells show significant reduction in both neuronal (5-fold) and astrocytic (13-fold) differentiation compared with those of control. These results clearly indicate that the blockage of gap junction-mediated intercellular communication interferes with differentiation of P19 cells into neurons and astrocytes.     

Investigating the safety factor at an invertebrate neuromuscular junction

Fidelity of synaptic transmission is essential at the neuromuscular junction (NMJ). To ensure that transmission does not fail, vertebrate motoneurons often release more neurotransmitter than is required for muscle contraction. This safety factor allows some loss of synaptic function without failure of muscle contraction. It is not known whether a similar mechanism operates at the invertebrate neuromuscular junction. In our study of the Drosophila NMJ, we find that glutamate receptor mutants can exhibit a substantial decrease in synaptic function while maintaining muscle contraction. The persistence of neuromuscular function in these mutants is not explained by synaptic facilitation, temporal summation of high frequency stimuli, or a hyperpolarizing shift in the activation range of muscle calcium channels. Instead, the attenuated synaptic response is sufficient to drive muscle contraction. Quantitative analysis of the decrease in synaptic transmission in these mutants implies that at the wild-type NMJ there is an approximately five- to ninefold excess in released transmitter. Hence, the presence of a synaptic safety factor is a conserved feature of neuromuscular organization in both invertebrates and vertebrates.     

Zinc inhibits apoptosis upstream of ICE/CED-3 proteases rather than at the level of an endonuclease

Apoptosis is commonly associated with DNA digestion, but it remains controversial as to which endonuclease is involved. The ability of zinc to inhibit DNA digestion in intact cells, and inhibit a Ca2+/Mg2+-dependent endonuclease in cell lysates, has been used frequently to suggest this is the endonuclease involved. However, zinc has many other effects on cells, and here it is shown that zinc also prevents many upstream events in apoptosis. These studies were performed in human ML-1 cells following incubation with etoposide. During apoptosis, these cells undergo intracellular acidification, increased accumulation of Hoechst 33342, DNA digestion and chromatin condensation. Zinc inhibited all of these events. An upstream event in apoptosis is activation of ICE/CED-3 proteases which is commonly observed as proteolysis of a substrate protein, poly(ADP-ribose) polymerase (PARP). The ICE/CED-3 proteases are themselves activated by proteolysis, and this was detected here by cleavage of one family member CPP32. Zinc prevented cleavage of both CPP32 and PARP. We recently demonstrated that dephosphorylation of the retinoblastoma susceptibility protein Rb was a marker of an event even further upstream in apoptosis; zinc was also found to inhibit Rb dephosphorylation. Therefore, zinc must protect cells at a very early step in the apoptotic pathway, and not as a direct inhibitor of an endonuclease.     

Coiled-coil unwinding at the smooth muscle myosin head-rod junction is required for optimal mechanical performance

Myosin II has two heads that are joined together by an alpha-helical coiled-coil rod, which can separate in the region adjacent to the head-rod junction (Trybus, K. M. 1994. J. Biol. Chem. 269:20819-20822). To test whether this flexibility at the head-rod junction is important for the mechanical performance of myosin, we used the optical trap to measure the unitary displacements of heavy meromyosin constructs in which a stable coiled-coil sequence derived from the leucine zipper was introduced into the myosin rod. The zipper was positioned either immediately after the heads (0-hep zip) or following 15 heptads of native sequence (15-hep zip). The unitary displacement (d) decreased from d = 9.7 +/- 0.6 nm for wild-type heavy meromyosin (WT HMM) to d = 0.1 +/- 0.3 nm for the 0-hep zip construct (mean +/- SE). Native values were restored in the 15-hep zip construct (d = 7.5 +/- 0.7 nm). We conclude that flexibility at the myosin head-rod junction, which is provided by an unstable coiled-coil region, is essential for optimal mechanical performance.     

Defining the optimal conditions for immunoperoxidase cytochemistry at the submicroscopic level]

The authors tested a number of experimental protocols and chemicals known to facilitate permeabilization of tissues to immunoperoxidase markers without ultrastructural alterations of the cells to be examined. Monoclonal antibodies producing hybridoma lymphomas served as a primary test object. None of the procedures employed (i.e., quenching of the fixative aldehydes by some reducing agents; cryopermeabilization; treatment by detergents) were shown either to intensify stainability or to increase the penetration of immunoreagents into the tissue depth. The diffusion efficiency depended only on the marker molecular mass and the thickness of the vibratome section incubated. The Elder and coworkers (1983) two-step technique has been found superior in the preservation of both immunoreactivity and fine structure of the cell.     

3'-Half of the thrombopoietin cDNA confers higher expression of erythropoietin at the RNA level but not at the protein level

Both erythropoietin (EPO) and the short-form thrombopoietin (TPO) were expressed at low levels whereas the long-form TPO was expressed at high levels in transgenic animals. To elucidate the role of carboxy-terminal half of the long-form TPO which is absent in the short-form, we generated recombinant TPO or EPO expression vectors which contain or lack the carboxy-terminal half of TPO and examined their expression in the HC11 and 293 cells. The long-form TPO was expressed higher than the short-form regardless of the cell types, transfection modes, and promoters. When 3'-half of the long-form TPO cDNA was placed downstream of the EPO cDNA to act as a 3'-untranslated region, expression of EPO was moderately increased at the RNA level, however, no remarkable increase was observed at the protein level. These results suggest that the low expression of EPO, as like as the short-form TPO, is due to absence of the 3'-half in the full-length TPO that confers stability both at the RNA and protein levels.