Role Of S1 Research Articles

Structural dynamics of protein S1 on the 70S ribosome visualized by ensemble cryo-EM.

Bacterial ribosomal protein S1 is the largest and highly flexible protein of the 30S subunit, and one of a few core ribosomal proteins for which a complete structure is lacking. S1 is thought to participate in transcription and translation. Best understood is the role of S1 in facilitating translation of mRNAs with structured 5' UTRs. Here, we present cryo-EM analyses of the 70S ribosome that reveal multiple conformations of S1. Based on comparison of several 3D maximum likelihood classification approaches in Frealign, we propose a streamlined strategy for visualizing a highly dynamic component of a large macromolecular assembly that itself exhibits high compositional and conformational heterogeneity. The resulting maps show how S1 docks at the ribosomal protein S2 near the mRNA exit channel. The globular OB-fold domains sample a wide area around the mRNA exit channel and interact with mobile tails of proteins S6 and S18. S1 also interacts with the mRNA entrance channel, where an OB-fold domain can be localized near S3 and S5. Our analyses suggest that S1 cooperates with other ribosomal proteins to form a dynamic mesh near the mRNA exit and entrance channels to modulate the binding, folding and movement of mRNA.

Investigation of exponential RNA amplification by the Qβ replicase complex

Positive-stranded RNA viruses are common among human pathogenic viruses, which often cooperate with host proteins to fulfill essential functions during infection. One function is replication of the viral genome. The Qβ phage is a positive-stranded RNA virus that infects E.coli. The Qβ replicase holo enzyme comprises the phage-encoded RNA-dependent RNA polymerase (β-subunit) and the host-encoded translation elongation factors, EF-Ts and EF-Tu as well as the ribosomal protein S1. The Qβ replicase has an extraordinary ability to exponentially amplify RNA in vivo and in vitro. A prerequisite for this is release of product and template RNA as single strands that can serve as new templates in subsequent rounds of replication. The role of S1 in the Qβ replicase is not clear. Recently, S1 was found to promote release of single-stranded product in Qβ replicase–mediated RNA synthesis. We have undertaken NMR spectroscopy and crystallization trials to improve our understanding of distinct S1 domains in solution as well as the ribosome- and replicase-binding properties of S1. Expression of distinct S1 domains for NMR spectroscopy has been optimized by use of autoinduction and results in high yields of [13C15N]-labelled protein fragments. These have proven very suitable for NMR studies and spectra revealed both ordered and disordered regions in the protein. Studies are ongoing. The structure of the Qβ core complex was recently determined at 2.5Å resolution. Thus, co-crystallization of the Qβ core in complex with S1 domains was undertaken and different crystal forms were obtained. These initial crystals diffracted to 3.2Å resolution and data processing as well as further optimization of the crystals is ongoing. S1 is thought to bind the β-subunit close to a region lined with basic amino acids, which potentially could facilitate interactions with the template RNA backbone and split it from the product strand. We demonstrate that neutralization of these basic amino acids indeed decrease or abolish infectivity of the Qβ phage. However, only one mutation, R503A affects the exponential replication in vitro. Crystallization of the Qβ holo enzyme bound to a truncated legitimate RNA template will be the next step for investigation of the mechanism of exponential RNA amplification by Qβ replicase.

Understanding Others' Feelings: The Role of the Right Primary Somatosensory Cortex in Encoding the Affective Valence of Others' Touch

Brain imaging studies in humans have shown the existence of a shared somatosensory representation in the primary somatosensory cortex (S1), putatively involved in understanding others' sensations (Keysers et al., 2010); however, the role of S1 in such a high-level process is still unknown. To ascertain the causal involvement of S1, and its possible hemispheric lateralization, in encoding the affective valence of emotional scenes, depicting, or not, a tactile event, we gave to healthy participants a picture-based affective go/no-go task and low-frequency repetitive transcranial magnetic stimulation (rTMS). The dorsolateral prefrontal cortex (DLPFC) was chosen as control site. rTMS over the right, but not the left, S1 selectively increased the participants' latencies in the affective go/no-go task, but only when the affective state was conveyed by touch; intriguingly, this interfering effect was associated with the empathic ability to adopt the subjective perspective of others. The left, not the right, DLPFC is also involved in affective go/no-go performance, but regardless of the sight of touch, and independently of empathic abilities. This novel evidence demonstrates the crossmodal role of right S1 in encoding the pleasant and aversive consequences of others' sensations evoked by touch.

A single missense mutation in a coiled-coil domain of Escherichia coli ribosomal protein S2 confers a thermosensitive phenotype that can be suppressed by ribosomal protein S1.

Ribosomal protein S2 is an essential component of translation machinery, and its viable mutated variants conferring distinct phenotypes serve as a valuable tool in studying the role of S2 in translation regulation. One of a few available rpsB mutants, rpsB1, shows thermosensitivity and ensures enhanced expression of leaderless mRNAs. In this study, we identified the nature of the rpsB1 mutation. Sequencing of the rpsB1 allele revealed a G-to-A transition in the part of the rpsB gene which encodes a coiled-coil domain of S2. The resulting E132K substitution resides in a highly conserved site, TKKE, a so-called N-terminal capping box, at the beginning of the second alpha helix. The protruding coiled-coil domain of S2 is known to provide binding with 16S rRNA in the head of the 30S subunit and, in addition, to interact with a key mRNA binding protein, S1. Molecular dynamics simulations revealed a detrimental impact of the E132K mutation on the coiled-coil structure and thereby on the interactions between S2 and 16S rRNA, providing a clue for the thermosensitivity of the rpsB1 mutant. Using a strain producing a leaderless lacZ transcript from the chromosomal lac promoter, we demonstrated that not only the rpsB1 mutation generating S2/S1-deficient ribosomes but also the rpsA::IS10 mutation leading to partial deficiency in S1 alone increased translation efficiency of the leaderless mRNA by about 10-fold. Moderate overexpression of S1 relieved all these effects and, moreover, suppressed the thermosensitive phenotype of rpsB1, indicating the role of S1 as an extragenic suppressor of the E132K mutation.

Association of Activity Changes in the Primary Sensory Cortex With Successful Motor Rehabilitation of the Hand Following Stroke

Background. Previous studies demonstrated a posterior shift of activation toward the primary sensory cortex (S1) following stroke; however, any relationship between this posterior shift and clinical outcome measures for the affected hand function were unclear. Objective. The authors investigated the possible role of S1 in motor recovery. Methods. Assuming that previous studies examined inhomogeneous groups of patients, the authors selected participants with chronic stroke who had moderate hand paresis, normal sensory examination and somatosensory-evoked potentials, and no lesion within the S1, thalamus, or brain stem. Constraint-induced movement therapy (CIMT) was used to train the impaired hand. To relate fMRI (functional MRI) activation changes from baseline to post-CIMT, a correlation analysis was performed with changes of the Wolf Motor Function Test (WMFT) as a test for the hand function. Results. A close relationship was found between increases in hand function and peak changes in activation within the ipsilesional S1. With a better outcome, greater increases in activation within the S1 were evident (P < .03; r = 0.73). Conclusion. In selected patients, the sensory network influences training-induced motor gains. This predictive knowledge of plasticity when applying CIMT may suggest strategies to enhance the effect of therapy, such as the addition of electrical stimulation to enhance S1 excitability.

COMPARISON OF PRIMARY AND SECONDARY SOMATOSENSORY CORTICAL RESPONSES TO FOREPAW STIMULATION

Two cortical regions, primary somatosensory (S1) and secondary somatosensory cortex (S2), are engaged in coding somatosensory information in all mammals. Although the role of S1 has been extensively reported, the anatomical and functional knowledge for S2 remained to be determined. Our aim was to investigate how S2 participates in information processing by comparing with corresponding S1 region. Ensemble neuronal activities of S1 and S2 forepaw regions were obtained by multiple-channel multiple single-unit recording method, and electrical stimulations of various intensities were applied to the forepaw. Several similarities between stimulation-evoked S2 and S1 neuronal responses were found. First, S2 neurons' activations were with similar onset and peak latencies in comparison with those of the S1 neurons at all intensities tested. Second, S2 and S1 neurons showed statistically indistinguishable half maximum duration. In addition, neurons in both areas responded to electrical stimulation with similar response magnitude. Finally, the response magnitude of S2 and S1 neurons showed comparable variation with increasing stimulation intensity. This similarity in response patterns between S1 and S2 neurons to peripheral stimulation suggested that S2 and S1 shared comparable hierarchy in anatomical and functional aspects in the somatosensory system.

COMPARISON OF PRIMARY AND SECONDARY SOMATOSENSORY CORTICAL RESPONSES TO FOREPAW STIMULATION

Two cortical regions, primary somatosensory (S1) and secondary somatosensory cortex (S2), are engaged in coding somatosensory information in all mammals. Although the role of S1 has been extensively reported, the anatomical and functional knowledge for S2 remained to be determined. Our aim was to investigate how S2 participates in information processing by comparing with corresponding S1 region. Ensemble neuronal activities of S1 and S2 forepaw regions were obtained by multiple-channel multiple single-unit recording method, and electrical stimulations of various intensities were applied to the forepaw. Several similarities between stimulation-evoked S2 and S1 neuronal responses were found. First, S2 neurons' activations were with similar onset and peak latencies in comparison with those of the S1 neurons at all intensities tested. Second, S2 and S1 neurons showed statistically indistinguishable half maximum duration. In addition, neurons in both areas responded to electrical stimulation with similar response magnitude. Finally, the response magnitude of S2 and S1 neurons showed comparable variation with increasing stimulation intensity. This similarity in response patterns between S1 and S2 neurons to peripheral stimulation suggested that S2 and S1 shared comparable hierarchy in anatomical and functional aspects in the somatosensory system.

Ribosomal Protein S1 Is not Essential for the trans-translation Machinery

In eubacteria, ribosome stalling during protein synthesis is rescued by a tmRNA-derived trans-translation system. Because ribosomal protein S1 specifically binds to tmRNA with high affinity, it is considered to be involved in the trans-translation system. However, the role of S1 in trans-translation is still unclear. To study the function of S1 in the trans-translation system, we constructed an S1-free cell-free translation system. We found that trans-translation proceeded even in the absence of S1. Addition of S1 into the S1-free system did not affect trans-translation efficiency. These results suggest that S1 does not play a role in the trans-translation machinery.

Proteolytic activities in two wood-decaying basidiomycete fungi, Serpula lacrymans and Coriolus versicolor

Summary: Proteolytic enzyme activities of the wood-decaying basidiomycetes Serpula lacrymans and Coriolus versicolor, have been characterized using azocasein as substrate and by electrophoretic analysis with gelatin-containing polyacrylamide gels (gelatin-SDS-PAGE). In S. lacrymans, intracellular and extracellular azocaseinase activity was optimal at pH 5-6 and was inhibited by pepstatin A. Gelatin-SDS-PAGE revealed two highly active proteinases, S1 and S4 (apparent M r 65000 and 30000, respectively) and two less active enzymes, S2 and S3 (apparent M r 47000 and 43000, respectively). S1, the predominant intracellular proteinase, was present at all ages of the mycelium (tested up to 3 months). It is active over a broad pH range, with highest activity around neutral pH. As S1 was partially inhibited by 1,10-phenanthroline, the enzyme was considered to be a metalloproteinase although EDTA and phosphoramidon had no effect. A proteinase apparently identical to S1 was also detected in the medium of older cultures. S4 is a pepstatin-sensitive aspartic proteinase; its activity was highly pH-dependent and it was inactive in gelatin gels at pH 5-0 and above. S2 and S3 were identified as intracellular metalloproteinases, present in relatively young and growing cultures. They were distinct from S1 as they were inhibited by EDTA and phosphoramidon. During starvation-induced autolysis of S. lacrymans, proteinase S1 was the only enzyme present throughout (and the intracellular azocaseinase activity increased), which suggested a likely role of S1 in intra-hyphal protein mobilization. S4 is more likely to play a part in extracellular digestion of protein. The azocaseinase activities of cultures of C. versicolor were optimal at pH 7-0 (intracellular) and pH 5-6 (extracellular). Mycelial extracts gave one major band of proteinase activity in gelatin gels, C1 (apparent M r 62-64000). Since the activity was sensitive to inhibitors of both serine and metalloproteinases, there may have been overlapping bands due to enzymes of both types. Extracellular samples gave a more complex pattern, (five bands, C2-C6, M r 50000-100000). C2 and C4 are PMSF-sensitive proteinases, C5 and C6 are probably metalloproteinases, while C3, which was most active at pH 4-0, was unaffected by any of the inhibitors tested, including pepstatin A. No aspartic proteinase equivalent to S. lacrymans S4 appeared to be produced by C. versicolor. From the information gained about the intracellular or extracellular location of these enzymes, and the conditions under which they are active, an in vivo role may be tentatively ascribed to some of them.

Nucleic acid helix-unwinding properties of ribosomal protein S1 and the role of S1 in mRNA binding to ribosomes.

The presence of ribosomal protein S1 in 30S ribosomes is indispensable for the formation of 30S initiation complexes with natural mRNA. The 30S subunits lacking S1 retain activity with AUG as mRNA and are also active in poly(rU)-directed binding of Phe-tRNA. Isolated protein S1 stoichiometrically disrupts the secondary structure of helical and stacked single-stranded polynucleotides and converts them into their fully or partially denatured forms. A mono-N-ethylmaleimide derivatives of S1 is nearly devoid of any RNA helix-unwinding properties but is readily incorporated into 30S subunits deficient in S1. The resulting N-ethylmaleimide-S1-containing 30S subunits are completely inactive in the binding of MS2 [3H]RNA and in the formation of an initiation complex with MS2 RNA as mRNA. They retain activity in the binding of the initiator fMet-tRNA in response to the trinucleotide AUG and in the binding of Phe-tRNA in response to poly(U). They also retain the capacity to bind 50S subunits and to form 70S couples. These results suggest that a correlation exists between the RNA helix-unwinding capacity of isolated S1 and the function of S1 in the ribosomal binding of natural mRNA when the protein becomes part of the 30S subunit.

Binding of ribosomal protein S1 of Escherichia coli to the 3' end of 16S rRNA.

Ribosomal protein S1 reversibly binds the 49-nucleotide fragment that is cleaved from the 3' end of 16S rRNA in ribosomes by colicin E3. The fragment has secondary structure in the form of a hairpin loop. At the base of the stem is a sequence (A-C-C-U-C-C) thought to be involved in the base pairing with complementary sequences in mRNA during the initiation of protein synthesis. The role of S1 may be to stabilize this region of the fragment in an open conformation to allow for base pairing to mRNA. This model is supported by the observation that S1 binds specifically to this region of the fragment. In addition, aurin tricarboxylic acid, an inhibitor of protein synthesis, reverses this effect by disrupting the S1-RNA complex. These results can explain why S1 is an essential component of the ribosome for translation of natural mRNA and why aurin tricarboxylic acid blocks initiation.