RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis

RsgA is a 30S ribosomal subunit-binding GTPase with an unknown function, shortage of which impairs maturation of the 30S subunit. We identified multiple gain-of-function mutants of Escherichia coli rbfA, the gene for a ribosome-binding factor, that suppress defects in growth and maturation of the 30S subunit of an rsgA-null strain. These mutations promote spontaneous release of RbfA from the 30S subunit, indicating that cellular disorders upon depletion of RsgA are due to prolonged retention of RbfA on the 30S subunit. We also found that RsgA enhances release of RbfA from the mature 30S subunit in a GTP-dependent manner but not from a precursor form of the 30S subunit. These findings indicate that the function of RsgA is to release RbfA from the 30S subunit during a late stage of ribosome biosynthesis. This is the first example of the action of a GTPase on the bacterial ribosome assembly described at the molecular level.     

Post-transcriptional regulation of ribosome accumulation during myoblast differentiation

The synthesis, accumulation and stability of rRNA were examined in embryonic quail myoblasts differentiating in cell culture. Quail myoblasts initially divide rapidly in culture, and accumulate 28S and 18S rRNA and ribosomes at a rate which maintains a constant ribosome content during cell division. After these myoblasts fuse, cell division ceases and ribosomes accumulate in fibers, but at a reduced rate which is only one fourth that in dividing myoblasts. Measurements of rRNA stability by 3H-methyl-methionine pulse-chase analysis show that 28S and 18S rRNA formed by fibers turn over with half-lives of 45 hr, and rRNA formed by myoblasts remains stable until fusion and then also turns over in fibers. Turnover of rRNA in fibers accounts for only half the reduction in ribosome accumulation following myoblast fusion. Measurements of the incorporation of 3H-adenosine into rRNA and ATP pools show that the rates of synthesis of rRNA precursor do not decrease after myoblast fuse, but half the rRNA molecules synthesized by fibers are degraded during processing. Degradation of rRNA during processing reduces the rate of formation of 28S and 18S rRNA, and together with rRNA turnover quantitatively accounts for the reduced rate of ribosome accumulation in fibers.     

Dynamical features of the Plasmodium falciparum ribosome during translation

Plasmodium falciparum, the mosquito-transmitted Apicomplexan parasite, causes the most severe form of human malaria. In the asexual blood-stage, the parasite resides within erythrocytes where it proliferates, multiplies and finally spreads to new erythrocytes. Development of drugs targeting the ribosome, the site of protein synthesis, requires specific knowledge of its structure and work cycle, and, critically, the ways they differ from those in the human host. Here, we present five cryo-electron microscopy (cryo-EM) reconstructions of ribosomes purified from P. falciparum blood-stage schizonts at sub-nanometer resolution. Atomic models were built from these density maps by flexible fitting. Significantly, our study has taken advantage of new capabilities of cryo-EM, in visualizing several structures co-existing in the sample at once, at a resolution sufficient for building atomic models. We have discovered structural and dynamic features that differentiate the ribosomes of P. falciparum from those of mammalian system. Prompted by the absence of RACK1 on the ribosome in our and an earlier study we confirmed that RACK1 does not specifically co-purify with the 80S fraction in schizonts. More extensive studies, using cryo-EM methodology, of translation in the parasite will provide structural knowledge that may lead to development of novel anti-malarials.     

Changes in the excitability of soleus muscle short latency stretch reflexes during human hopping after 4 weeks of hopping training

Changes in the excitability of the human triceps surae muscle short latency stretch reflexes were investigated in six male subjects before and after 4 weeks of progressive two-legged hopping training. During the measurements the subjects performed 2-Hz hopping with: preferred contact time (PCT) and short contact time. The following reflex parameters were examined before and after the training period: the soleus muscle (SOL) Hoffmann-reflex (H-reflex) at rest and during hopping, the short latency electromyogram (EMG) components of the movement induced stretch reflex (MSR) in SOL and medial gastrocnemius muscle (MG), and the EMG amplitude of the SOL and MG tendon reflexes (T-reflexes) elicited at rest. The main results can be summarized as follows: the SOL T-reflex had increased by about 28% (P < 0.05) after training while the MG T-reflex was unchanged; the SOL MSR (always evident) and the MG MSR (when observable) did not change in amplitude with training, and before training the SOL H-reflex in both hopping situations was significantly depressed to about 40% of the reference value at standing rest (P < 0.05). After training the H-reflex during PCT hopping was no longer depressed. As the value of the measured mechanical parameters (the total work rate, joint angular velocity and the ankle joint work rate) was unchanged after training in both hopping situations, the reflex changes observed could not be ascribed to changes in the movement pattern. To explain the observed changes, hypotheses of changes in the excitability of the stretch reflex caused by the training were taken into consideration and discussed. Accepted: 22 May 1998     

Structural changes in the ribosome during the elongation cycle

Ample data on structural changes that arise in the ribosome during translation have been accumulated. The most interesting information on such changes has been obtained by cryoelectron microscopy of ribosome complexes with various ligands and by rRNA site-directed mutagenesis combined with a structural analysis of the ribosome by a chemical modification technique (chemical probing). The review considers the best-known structural changes that arise in the translating ribosome upon its interactions with tRNA and the elongation factors. The changes are discussed in the context of interactions between the functional centers of the ribosome. A universal system of rRNA helices and proteins is described in detail. The system integrates the functional centers of the ribosome and allows transduction of allosteric conformational signals. Biochemical data are considered in terms of the structures and interactions of ribosomal elements, and a hypothesis is advanced that the position of the GTPase-associated center in the ribosome regulates the binding of the elongation factors.     

Neural control of leg stiffness during hopping in boys and men

The purpose of the study was to investigate whether boys and men utilise different control strategies whilst hopping. Eleven boys (11–12 yr old) and ten men completed hopping at 1.5 Hz, 3.0 Hz and at their preferred frequency. A footswitch measured contact and flight times, from which leg stiffness was calculated. Simultaneously, surface electromyograms (EMGs) of selected lower limb muscles were recorded and quantified for each 30 ms period during the first 120 ms post-ground contact. At 1.5 Hz there were no differences between the groups in relative stiffness or muscle activity. At 3.0 Hz men had significantly shorter contact times (P = 0.013), longer flight times (P = 0.002), greater relative stiffness (P = 0.01) and significantly greater soleus (P = 0.012) and vastus lateralis (P < 0.001) activity during the initial 30 ms post-ground contact. At the preferred frequency men hopped significantly faster than the boys (P = 0.007), with greater leg stiffness (P < 0.01) and with more extensor activity in most time periods. Boys and men demonstrated similar control strategies when hopping at a slow frequency, but when hopping frequency increased men were able to better increase feedforward and reflex muscle activity to hop with greater relative stiffness.     

An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue

In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon–anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB''s affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA.     

Potential roles for ubiquitin and the proteasome during ribosome biogenesis

We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.     

The structural changes in the ribosome during the elongation cycle

The plenty of data about structural changes in the ribosome during its functioning has been accumulated. The most interesting information on such changes was obtained by cryo-EM of various ribosomal complexes with the ligands and by combination of rRNA site-directed mutagenesis with the analysis of structural changes in ribosome by chemical modification technique (chemical probing). The most studied structural transformations of the ribosome interacting with tRNAs and elongation factors are considered in this review. The structural rearrangements are discussed in the context of interactions between the functional centers of the ribosome. We also describe the system of tertiary contacts between the rRNA helices and proteins which forms the universal structure in the ribosome. We pay attention that by means of such system the allosteric conformational signal can be transmitted between the functional centers. Besides the discussion of different biochemical data in the scope of structural data we also consider the hypothesis that the position of GTPase associated center (GAC) in the ribosome regulates the binding of elongation factors.     

Knee stiffness is a major determinant of leg stiffness during maximal hopping

Understanding stiffness of the lower extremities during human movement may provide important information for developing more effective training methods during sports activities. It has been reported that leg stiffness during submaximal hopping depends primarily on ankle stiffness, but the way stiffness is regulated in maximal hopping is unknown. The goal of this study was to examine the hypothesis that knee stiffness is a major determinant of leg stiffness during the maximal hopping. Ten well-trained male athletes performed two-legged hopping in place with a maximal effort. We determined leg and joint stiffness of the hip, knee, and ankle from kinetic and kinematic data. Knee stiffness was significantly higher than ankle and hip stiffness. Further, the regression model revealed that only knee stiffness was significantly correlated with leg stiffness. The results of the present study suggest that the knee stiffness, rather than those of the ankle or hip, is the major determinant of leg stiffness during maximal hopping.     

Leg stiffness can be maintained during reactive hopping despite modified acceleration conditions

AimThe aim of the present study was to evaluate reactive hops under systematically modified acceleration conditions. It was hypothesized that a high preactivity of the leg extensors and phase-specific adjustments of the leg muscle activation would compensate the alterations caused by the various acceleration levels in order to maintain a high leg stiffness, thus enabling the jumper to perform truly reactive jumps with short ground contact times despite the unaccustomed acceleration conditions.MethodsGround reaction forces (GRF), kinematic and electromyographic data of 20 healthy subjects were recorded during reactive hopping in a special sledge jump system for seven different acceleration levels: three acceleration levels with lower than normal gravity (0.7g, 0.8g, 0.9g), one with gravitational acceleration (1g) and three with higher acceleration (1.1g, 1.2g, 1.3g).ResultsThe increase of the acceleration from 0.7g to 1.3g had no significant effect on the preactivity of the leg extensors, the leg stiffness and the rate of force development. However, it resulted in increased peak GRF (+15%), longer ground contact time (+10%) and increased angular excursion at the ankle and knee joints (+3°).DiscussionThroughout a wide acceleration range, the subjects were able to maintain a high leg stiffness and perform reactive hops by keeping the preactivity constantly high and adjusting the muscle activity in the later phases. In consequence, it can be concluded that the neuromuscular system can cope with different acceleration levels, at least in the acceleration range used in this study.     

Initiation of ribosome degradation during starvation in Escherichia coli

Ribosomes are known to be degraded under conditions of nutrient limitation. However, the mechanism by which a normally stable ribosome becomes a substrate for the degradation machinery has remained elusive. Here, we present in vitro and in vivo data demonstrating that free ribosome subunits are the actual targets of the degradative enzymes, whereas 70S particles are protected from such degradation. Conditions that increase the formation of subunits both in vitro and in vivo lead to enhanced degradation, while conditions favoring the presence of intact 70S ribosomes prevent or reduce breakdown. Thus, the simple formation of free 50S and 30S subunits is sufficient to serve as the initiation mechanism that allows endoribonuclease cleavage and subsequent ribosome breakdown.     

A motion analysis marker-based method of determining centre of pressure during two-legged hopping

The fixed position of force plates has led researchers to pursue alternative methods of determining centre of pressure (CoP) location. To date, errors reported using alternative methods to the force plate during dynamic tasks have been high. The aim of this study was to investigate the accuracy of a motion analysis marker-based system to determine CoP during a two-legged hopping task. Five markers were attached to the left and right feet of eight healthy adults (5 females, 3 males, age: 25.0±2.8 years, height: 1.75±0.07 m, mass: 71.3±11.3 kg). Multivariate forward stepwise and forced entry linear regression was used with data from five participants to determine CoP position during quiet standing and hopping at various frequencies. Maximum standard error of the estimate of CoP position was 12 mm in the anteroposterior direction and 8 mm in the mediolateral. Cross-validation was performed using the remaining 3 participants. Maximum root mean square difference between the force plate and marker method was 14 mm for mediolateral CoP and 20 mm for anteroposterior CoP during 1.5 Hz hopping. Differences reduced to a maximum of 7 mm (mediolateral) and 14 mm (anteroposterior) for the other frequencies. The smallest difference in calculated sagittal plane ankle moment and timing of maximum moment was during 3.0 Hz hopping, and largest at 1.5 Hz. Results indicate the marker-based method of determining CoP may be a suitable alternative to a force plate to determine CoP position during a two-legged hopping task at frequencies greater than 1.5 Hz.     

Age- and gender-related development of stretch shortening cycle during a sub-maximal hopping task

The aim of this study was to analyse the effects of age and gender (and their interaction) on a stretch shortening cycle solicited during a hopping task. For this aim, 147 girls and 148 boys aged 11 to 20 years, who were enrolled in middle school or secondary school with no experience in sport activity, or training less than three times per week, performed 3×5 hops in place. Leg-stiffness, jump-height and reactive-strength indices were assessed using an accelerometer (Myotest). The participants were selected in order to form five age groups: 11 12, 13-14, 15-16, 17-18 and 19-20 years. Regression analysis between force and centre of mass displacement revealed spring-mass behaviour for all groups (r²=.73-.89), meaning that beginning at the age of 11 years, children are able to perform complex inter-muscular coordination of the lower limbs, revealing efficient neural control early in childhood. Leg stiffness increased from 24.7 ± 10.6 kN · m⁻¹ at 11-12 years to 44.1 ± 14 kN · m⁻¹ in boys, with a small increase until 16 years (+17%) and a large increase between 17 and 20 years (+32.7%). In girls, leg stiffness increased from 26.6 ± 9 kN · m⁻¹ at 11-12 years to 39.4 ± 10.9 kN · m⁻¹ at 19-20 years, with a curious decrease in leg stiffness at 17-18 years, probably due to an increase in the percentage of fat at this age (25%). While no gender effect was found, the reactive-strength index revealed that, from 15-16 years onward, boys were better able to produce high levels of force in a shorter time than girls. The age of 15-16 years is a threshold of maturity and gender differentiation, where the boys investigated are more efficient in the stretch shortening cycle.