Translation of non-capped mRNAs in a eukaryotic cell-free system: acceleration of initiation rate in the course of polysome formation

Real-time monitoring of the translation of non-capped luciferase mRNA in a wheat germ cell-free system has been performed by continuous in situ measurement of the luminescence increase in the translation mixture. The phenomenon of acceleration of translation has been revealed. It has been shown that the acceleration is accompanied by the loading of translating polysomes with additional ribosomes, and thus is caused mainly by a rise in the initiation rate, rather than the stimulation of elongation or the involvement of additional mRNA molecules in translation. The acceleration requires a sufficient concentration of mRNA and depends on the sequence of the 5′ untranslated region (UTR). It can be abolished by the addition of excess cap analog (m7GpppGm). As the acceleration does not depend on the preliminary translation of other mRNAs in the same extract, the conclusion has been made that the effect is not due to activation of the ribosome population or other components of the system during translation, but rather it is the consequence of intra-polysomal events. The acceleration observed is discussed in terms of the model of two overlapping initiation pathways in eukaryotic polysomes: translation of non-capped mRNAs starts with eIF4F-independent initiation at 5′ UTR, and after the formation of sufficiently loaded polysomes, they rearrange in such a way that a mechanism of re-initiation of terminating ribosomes switches on. The eIF4F-mediated circularization of polysomes may be considered as a possible event that leads to the re-initiation switch and the resultant acceleration effect

Unidirectional constant rate motion of the ribosomal scanning particle during eukaryotic translation initiation

According to the model of translation initiation in eukaryotes, the 40S ribosomal subunit binds to capped 5′-end of mRNA and subsequently migrates along 5′-UTR in searching for initiation codon. However, it remains unclear whether the migration is the result of a random one-dimensional diffusion, or it is an energy-driven unidirectional movement. To address this issue, the method of continuous monitoring of protein synthesis in situ was used for high precision measurements of the times required for translation of mRNA with 5′-UTRs of different lengths and structures in mammalian and plant cell-free systems. For the first time, the relationship between the scanning time and the 5′-UTR length was determined and their linear correlation was experimentally demonstrated. The conclusion is made that the ribosome migration is an unidirectional motion with the rate being virtually independent of a particular mRNA sequence and secondary structure

Cysteine residues 244 and 458-459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions

Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD) and nucleotide binding domain (NBD) of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines' thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG). Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459) were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor function of the Na,K-ATPase and alter responses of the enzyme to hypoxia or upon treatment with cardiotonic steroids

Anomalous self-diffusion in the ferromagnetic Ising chain with Kawasaki dynamics

We investigate the motion of a tagged spin in a ferromagnetic Ising chain evolving under Kawasaki dynamics. At equilibrium, the displacement is Gaussian, with a variance growing as $A t^{1/2}$. The temperature dependence of the prefactor $A$ is derived exactly. At low temperature, where the static correlation length $\xi$ is large, the mean square displacement grows as $(t/\xi^2)^{2/3}$ in the coarsening regime, i.e., as a finite fraction of the mean square domain length. The case of totally asymmetric dynamics, where $(+)$ (resp. $(-)$) spins move only to the right (resp. to the left), is also considered. In the steady state, the displacement variance grows as $B t^{2/3}$. The temperature dependence of the prefactor $B$ is derived exactly, using the Kardar-Parisi-Zhang theory. At low temperature, the displacement variance grows as $t/\xi^2$ in the coarsening regime, again proportionally to the mean square domain length.Comment: 22 pages, 8 figures. A few minor changes and update

Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA

The time course of polysome formation was studied in a long-term wheat germ cell-free translation system using sedimentation and electron microscopy techniques. The polysomes were formed on uncapped luciferase mRNA with translation-enhancing 5′ and 3′ UTRs. The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases. First, short linear polysomes containing no more than six ribosomes were formed. Next, folding of these polysomes into short double-row clusters occurred. Subsequent gradual elongation of the clusters gave rise to heavy-loaded double-row strings containing up to 30–40 ribosomes. The formation of the double-row polysomes was considered to be equivalent to circularization of polysomes, with antiparallel halves of the circle being laterally stuck together by ribosome interactions. A slow exchange with free ribosomes and free mRNA observed in the double-row type polysomes, as well as the resistance of translation in them to AMP-PNP, provided evidence that most polysomal ribosomes reinitiate translation within the circularized polysomes without scanning of 5′ UTR, while de novo initiation including 5′ UTR scanning proceeds at a much slower rate. Removal or replacements of 5′ and 3′ UTRs affected the initial phase of translation, but did not prevent the formation of the double-row polysomes during translation

Nucleic Acids Res

Using sedimentation and cryo electron tomography techniques, the conformations of eukaryotic polyribosomes formed in a long-term cell-free translation system were analyzed over all the active system lifetime (20-30 translation rounds during 6-8 h in wheat germ extract at 25 degrees C). Three distinct types of the conformations were observed: (i) circular polyribosomes, varying from ring-shaped forms to circles collapsed into double rows, (ii) linear polyribosomes, tending to acquire planar zigzag-like forms and (iii) densely packed 3D helices. At the start, during the first two rounds of translation mostly the circular (ring-shaped and double-row) polyribosomes and the linear (free-shaped and zigzag-like) polyribosomes were formed ('juvenile phase'). The progressive loading of the polyribosomes with translating ribosomes induced the opening of the circular polyribosomes and the transformation of a major part of the linear polyribosomes into the dense 3D helices ('transitional phase'). After 2 h from the beginning (about 8-10 rounds of translation) this compact form of polyribosomes became predominant, whereas the circular and linear polyribosome fractions together contained less than half of polysomal ribosomes ('steady-state phase'). The latter proportions did not change for several hours. Functional tests showed a reduced translational activity in the fraction of the 3D helical polyribosomes

Nucleic Acids Res

The polyribosomes newly formed on recombinant GFP-encoding mRNAs in a wheat germ cell-free translation system were analyzed using cryo-electron tomography, with sub-tomogram averaging of polysomal ribosomes and reconstruction of 3D structures of individual polyribosomes. The achieved level of resolution in the reconstructed polyribosomes allowed deducing the mRNA path by connecting adjacent exit and entry sites at the ribosomes inside each polyribosome. In this way, the circularity of a significant fraction (about 50%) of translating polyribosomes was proved in the case of the capped poly(A)-tailed mRNA, in agreement with the existing paradigm of the circularization via interaction of cap-bound initiation factor eIF4F with poly(A)-binding protein. However, translation of the capped mRNA construct without poly(A) tail, but with unspecific 3'-UTR derived from non-coding plasmid sequence, also led to the formation of circular polyribosomes in similar proportion (40%). Moreover, the polyribosomes formed on the uncapped non-polyadenylated mRNA with non-synergistic 5'- and 3'-UTRs proved to be circular as well, and appeared in the same proportion as in the previous cases. Thus, the formation of circular polyribosomes was found to be virtually independent of the presence of cap structure and poly(A) tail in mRNA, in contrast to the longstanding paradigm in the field

Is Thermosensing Property of RNA Thermometers Unique?

A large number of studies have been dedicated to identify the structural and sequence based features of RNA thermometers, mRNAs that regulate their translation initiation rate with temperature. It has been shown that the melting of the ribosome-binding site (RBS) plays a prominent role in this thermosensing process. However, little is known as to how widespread this melting phenomenon is as earlier studies on the subject have worked with a small sample of known RNA thermometers. We have developed a novel method of studying the melting of RNAs with temperature by computationally sampling the distribution of the RNA structures at various temperatures using the RNA folding software Vienna. In this study, we compared the thermosensing property of 100 randomly selected mRNAs and three well known thermometers - rpoH, ibpA and agsA sequences from E. coli. We also compared the rpoH sequences from 81 mesophilic proteobacteria. Although both rpoH and ibpA show a higher rate of melting at their RBS compared with the mean of non-thermometers, contrary to our expectations these higher rates are not significant. Surprisingly, we also do not find any significant differences between rpoH thermometers from other -proteobacteria and E. coli non-thermometers

Quantitative analysis of ribosome–mRNA complexes at different translation stages

Inhibition of primer extension by ribosome–mRNA complexes (toeprinting) is a proven and powerful technique for studying mechanisms of mRNA translation. Here we have assayed an advanced toeprinting approach that employs fluorescently labeled DNA primers, followed by capillary electrophoresis utilizing standard instruments for sequencing and fragment analysis. We demonstrate that this improved technique is not merely fast and cost-effective, but also brings the primer extension inhibition method up to the next level. The electrophoretic pattern of the primer extension reaction can be characterized with a precision unattainable by the common toeprint analysis utilizing radioactive isotopes. This method allows us to detect and quantify stable ribosomal complexes at all stages of translation, including initiation, elongation and termination, generated during the complete translation process in both the in vitro reconstituted translation system and the cell lysate. We also point out the unique advantages of this new methodology, including the ability to assay sites of the ribosomal complex assembly on several mRNA species in the same reaction mixture

Identifying protein complexes directly from high-throughput TAP data with Markov random fields

<p>Abstract</p> <p>Background</p> <p>Predicting protein complexes from experimental data remains a challenge due to limited resolution and stochastic errors of high-throughput methods. Current algorithms to reconstruct the complexes typically rely on a two-step process. First, they construct an interaction graph from the data, predominantly using heuristics, and subsequently cluster its vertices to identify protein complexes.</p> <p>Results</p> <p>We propose a model-based identification of protein complexes directly from the experimental observations. Our model of protein complexes based on Markov random fields explicitly incorporates false negative and false positive errors and exhibits a high robustness to noise. A model-based quality score for the resulting clusters allows us to identify reliable predictions in the complete data set. Comparisons with prior work on reference data sets shows favorable results, particularly for larger unfiltered data sets. Additional information on predictions, including the source code under the GNU Public License can be found at http://algorithmics.molgen.mpg.de/Static/Supplements/ProteinComplexes.</p> <p>Conclusion</p> <p>We can identify complexes in the data obtained from high-throughput experiments without prior elimination of proteins or weak interactions. The few parameters of our model, which does not rely on heuristics, can be estimated using maximum likelihood without a reference data set. This is particularly important for protein complex studies in organisms that do not have an established reference frame of known protein complexes.</p