Although trans-translation mediated by tmRNA-SmpB has long been known as the sole system to relieve bacterial stalled ribosomes, ArfA has recently been identified as an alternative factor for ribosome rescue in Escherichia coli. This process requires hydrolysis of nascent peptidyl-tRNA by RF2, which usually acts as a stop codon-specific peptide release factor. It poses a fascinating question of how ArfA and RF2 recognize and rescue the stalled ribosome. Here, we mapped the location of ArfA in the stalled ribosome by directed hydroxyl radical probing. It revealed an ArfA-binding site around the neck region of the 30S subunit in which the N- and C-terminal regions of ArfA are close to the decoding center and the mRNA entry channel, respectively. ArfA and RF2 sequentially enter the ribosome stalled in either the middle or 3' end of mRNA, whereas RF2 induces a productive conformational change of ArfA only when ribosome is stalled at the 3' end of mRNA. On the basis of these results, we propose that ArfA functions as the sensor to recognize the target ribosome after RF2 binding.
Messenger RNAs lacking a stop codon trap ribosomes at their 3' ends, depleting the pool of ribosomes available for protein synthesis. In bacteria, a remarkable quality control system rescues and recycles stalled ribosomes in a process known as trans-translation. Acting as a tRNA, transfer-messenger RNA (tmRNA) is aminoacylated, delivered by EF-Tu to the ribosomal A site, and accepts the nascent polypeptide. Translation then resumes on a reading frame within tmRNA, encoding a short peptide tag that targets the nascent peptide for degradation by proteases. One unsolved issue in trans-translation is how tmRNA and its protein partner SmpB preferentially recognize stalled ribosomes and not actively translating ones. Here, we examine the effect of the length of the 3' extension of mRNA on each step of trans-translation by pre-steady-state kinetic methods and fluorescence polarization binding assays. Unexpectedly, EF-Tu activation and GTP hydrolysis occur rapidly regardless of the length of the mRNA, although the peptidyl transfer to tmRNA decreases as the mRNA 3' extension increases and the tmRNA·SmpB binds less tightly to the ribosome with an mRNA having a long 3' extension. From these results, we conclude that the tmRNA·SmpB complex dissociates during accommodation due to competition between the downstream mRNA and the C-terminal tail for the mRNA channel. Rejection of the tmRNA·SmpB complex during accommodation is reminiscent of the rejection of near-cognate tRNA from the ribosome in canonical translation.
Small regulatory RNAs (sRNAs) are conserved among a wide range of bacteria. They modulate the translational efficiency of target mRNAs through base-pairing with the help of RNA chaperone Hfq. The present study identified a novel sRNA, Esr41 (enterohemorrhagic Escherichia coli O157 small RNA #41), from an intergenic region of an enterohemorrhagic E. coli (EHEC) O157:H7 Sakai-specific sequence that is not present in the nonpathogenic E. coli K-12. Esr41 was detected as an RNA molecule approximately 70 nucleotides long with a 3' GC-rich palindrome sequence followed by a long poly(U), which is a characteristic of rho-independent terminators and is also a structural feature required for the action of Hfq. EHEC O157 harboring a multicopy plasmid carrying the esr41 gene increased cell motility and the expression of fliC, a gene encoding a major flagellar component. These results indicate that Esr41 stimulates fliC expression in EHEC O157. Furthermore, the increase in cell motility induced by Esr41 was also observed in the E. coli K-12, suggesting that target genes controlled by Esr41 are present in both EHEC O157 and K-12.
The in vivo assembly of ribosomal subunits is a highly complex process, with a tight coordination between protein assembly and rRNA maturation events, such as folding and processing of rRNA precursors, as well as modifications of selected bases. In the cell, a large number of factors are required to ensure the efficiency and fidelity of subunit production. Here we characterize the immature 30S subunits accumulated in a factor-null Escherichia coli strain (?rsgA?rbfA). The immature 30S subunits isolated with varying salt concentrations in the buffer system show interesting differences on both protein composition and structure. Specifically, intermediates derived under the two contrasting salt conditions (high and low) likely reflect two distinctive assembly stages, the relatively early and late stages of the 3' domain assembly, respectively. Detailed structural analysis demonstrates a mechanistic coupling between the maturation of the 5' end of the 17S rRNA and the assembly of the 30S head domain, and attributes a unique role of S5 in coordinating these two events. Furthermore, our structural results likely reveal the location of the unprocessed terminal sequences of the 17S rRNA, and suggest that the maturation events of the 17S rRNA could be employed as quality control mechanisms on subunit production and protein translation.
Mutations in the human CACNA1F gene cause incomplete congenital stationary night blindness type 2 (CSNB2), a non-progressive, clinically heterogeneous retinal disorder. However, the molecular mechanisms underlying CSNB2 have not been fully explored. Here, we describe the positional cloning of a blind zebrafish mutant, wait until dark (wud), which encodes a zebrafish homolog of human CACNA1F. We identified two zebrafish cacna1f paralogs and showed that the cacna1fa transcript (the gene mutated in wud) is expressed exclusively in the photoreceptor layer. We demonstrated that Cacna1fa localizes at the photoreceptor synapse and is absent from wud mutants. Electroretinograms revealed abnormal cone photoreceptor responses from wud mutants, indicating a defect in synaptic transmission. Although there are no obvious morphological differences, we found that wud mutants lacked synaptic ribbons and that wud is essential for the development of synaptic ribbons. We found that Ribeye, the most prominent synaptic ribbon protein, was less abundant and mislocalized in adult wud mutants. In addition to cloning wud, we identified synaptojanin 1 (synj1) as the defective gene in slacker (slak), a blind mutant with floating synaptic ribbons. We determined that Cacna1fa was expressed in slak photoreceptors and that Synj1 was initially expressed wud photoreceptors, but was absent by 5 days postfertilization. Collectively, our data demonstrate that Cacna1fa is essential for cone photoreceptor function and synaptic ribbon formation and reveal a previously unknown yet critical role of L-type voltage-dependent calcium channels in the expression and/or distribution of synaptic ribbon proteins, providing a new model to study the clinical variability in human CSNB2 patients.
The tRNA gene data base curated by experts "tRNADB-CE" (http://trna.ie.niigata-u.ac.jp) was constructed by analyzing 1,966 complete and 5,272 draft genomes of prokaryotes, 171 viruses', 121 chloroplasts', and 12 eukaryotes' genomes plus fragment sequences obtained by metagenome studies of environmental samples. 595,115 tRNA genes in total, and thus two times of genes compiled previously, have been registered, for which sequence, clover-leaf structure, and results of sequence-similarity and oligonucleotide-pattern searches can be browsed. To provide collective knowledge with help from experts in tRNA researches, we added a column for enregistering comments to each tRNA. By grouping bacterial tRNAs with an identical sequence, we have found high phylogenetic preservation of tRNA sequences, especially at the phylum level. Since many species-unknown tRNAs from metagenomic sequences have sequences identical to those found in species-known prokaryotes, the identical sequence group (ISG) can provide phylogenetic markers to investigate the microbial community in an environmental ecosystem. This strategy can be applied to a huge amount of short sequences obtained from next-generation sequencers, as showing that tRNADB-CE is a well-timed database in the era of big sequence data. It is also discussed that batch-learning self-organizing-map with oligonucleotide composition is useful for efficient knowledge discovery from big sequence data.
Transfer messenger RNA (tmRNA; also known as 10Sa RNA or SsrA RNA) is a small RNA molecule that is conserved among bacteria. It has structural and functional similarities to tRNA: it has an upper half of the tRNA-like structure, its 5' end is processed by RNase P, it has typical tRNA-specific base modifications, it is aminoacylated with alanine, it binds to EF-Tu after aminoacylation and it enters the ribosome with EF-Tu and GTP. However, tmRNA lacks an anticodon, and instead it has a coding sequence for a short peptide called tag-peptide. An elaborate interplay of actions of tmRNA as both tRNA and mRNA with the help of a tmRNA-binding protein, SmpB, facilitates trans-translation, which produces a single polypeptide from two mRNA molecules. Initially alanyl-tmRNA in complex with EF-Tu and SmpB enters the vacant A-site of the stalled ribosome like aminoacyl-tRNA but without a codon-anticodon interaction, and subsequently truncated mRNA is replaced with the tag-encoding region of tmRNA. During these processes, not only tmRNA but also SmpB structurally and functionally mimics both tRNA and mRNA. Thus trans-translation rescues the stalled ribosome, thereby allowing recycling of the ribosome. Since the tag-peptide serves as a target of AAA(+) proteases, the trans-translation products are preferentially degraded so that they do not accumulate in the cell. Although alternative rescue systems have recently been revealed, trans-translation is the only system that universally exists in bacteria. Furthermore, it is unique in that it employs a small RNA and that it prevents accumulation of non-functional proteins from truncated mRNA in the cell. It might play the major role in rescuing the stalled translation in the bacterial cell.
tmRNA is a bacterial small RNA having a structure resembling the upper half of tRNA and its 3' end accepts alanine followed by binding to EF-Tu like tRNA. Instead of lacking a lower half of the cloverleaf structure including the anticodon, tmRNA has a short coding sequence for tag-peptide that serves as a target of cellular proteases. An elaborate coordination of two functions as tRNA and mRNA facilitates an irregular translation termed trans-translation: a single polypeptide is synthesized from two mRNA molecules. It allows resumption of translation stalled on a truncated mRNA, producing a chimeric polypeptide comprising the C-terminally truncated polypeptide derived from truncated mRNA and the C-terminal tag-peptide encoded by tmRNA. Trans-translation promotes recycling of the stalled ribosomes in the cell, and the resulting C-terminally tagged polypeptide is preferentially degraded by cellular proteases. Biochemical studies using in vitro trans-translation systems together with structural studies have unveiled the molecular mechanism of trans-translation, during which the upper and lower halves of tRNA are mimicked by the tRNA-like structure of tmRNA and a tmRNA-specific binding protein called SmpB, respectively. They mimic not only the tRNA structure but also its behavior perhaps at every step of the trans-translation process in the ribosome. Furthermore, the C-terminal tail of SmpB, which is unstructured in solution, occupies the mRNA path in the ribosome to play a crucial role in trans-translation, addressing how tmRNA·SmpB recognizes the ribosome stalled on a truncated mRNA.
The ribosome is an RNA- and protein-based macromolecule having multiple functional domains to facilitate protein synthesis, and it is synthesized through multiple steps including transcription, stepwise cleavages of the primary transcript, modifications of ribosomal proteins and RNAs and assemblies of ribosomal proteins with rRNAs. This process requires dozens of trans-acting factors including GTP- and ATP-binding proteins to overcome several energy-consuming steps. Despite accumulation of genetic, biochemical and structural data, the entire process of bacterial ribosome synthesis remains elusive. Here, we review GTPases involved in bacterial ribosome maturation.
To understand how the brain perceives the external world, it is desirable to observe neuronal activity in the brain in real time during perception. The zebrafish is a suitable model animal for fluorescence imaging studies to visualize neuronal activity because its body is transparent through the embryonic and larval stages. Imaging studies have been carried out to monitor neuronal activity in the larval spinal cord and brain using Ca(2+) indicator dyes and DNA-encoded Ca(2+) indicators, such as Cameleon, GFP-aequorin, and GCaMPs. However, temporal and spatial resolution and sensitivity of these tools are still limited, and imaging of brain activity during perception of a natural object has not yet been demonstrated. Here we demonstrate visualization of neuronal activity in the optic tectum of larval zebrafish by genetically expressing the new version of GCaMP. First, we demonstrate Ca(2+) transients in the tectum evoked by a moving spot on a display and identify direction-selective neurons. Second, we show tectal activity during perception of a natural object, a swimming paramecium, revealing a functional visuotopic map. Finally, we image the tectal responses of a free-swimming larval fish to a paramecium and thereby correlate neuronal activity in the brain with prey capture behavior.
The Rice Annotation Project Database (RAP-DB, http://rapdb.dna.affrc.go.jp/) has been providing a comprehensive set of gene annotations for the genome sequence of rice, Oryza sativa (japonica group) cv. Nipponbare. Since the first release in 2005, RAP-DB has been updated several times along with the genome assembly updates. Here, we present our newest RAP-DB based on the latest genome assembly, Os-Nipponbare-Reference-IRGSP-1.0 (IRGSP-1.0), which was released in 2011. We detected 37,869 loci by mapping transcript and protein sequences of 150 monocot species. To provide plant researchers with highly reliable and up to date rice gene annotations, we have been incorporating literature-based manually curated data, and 1,626 loci currently incorporate literature-based annotation data, including commonly used gene names or gene symbols. Transcriptional activities are shown at the nucleotide level by mapping RNA-Seq reads derived from 27 samples. We also mapped the Illumina reads of a Japanese leading japonica cultivar, Koshihikari, and a Chinese indica cultivar, Guangluai-4, to the genome and show alignments together with the single nucleotide polymorphisms (SNPs) and gene functional annotations through a newly developed browser, Short-Read Assembly Browser (S-RAB). We have developed two satellite databases, Plant Gene Family Database (PGFD) and Integrative Database of Cereal Gene Phylogeny (IDCGP), which display gene family and homologous gene relationships among diverse plant species. RAP-DB and the satellite databases offer simple and user-friendly web interfaces, enabling plant and genome researchers to access the data easily and facilitating a broad range of plant research topics.
Deciphering the genetic code is a fundamental process in all living organisms. In many bacteria, AUA codons are deciphered by tRNA(Ile2) bearing lysidine (L) at the wobble position. L is a modified cytidine introduced post-transcriptionally by tRNA(Ile)-lysidine synthetase (TilS). Some bacteria, including Mycoplasma mobile, do not carry the tilS gene, indicating that they have established a different system to decode AUA codons. In this study, tRNA(Ile2) has been isolated from M. mobile and was found to contain a UAU anticodon without any modification. Mycoplasma mobile isoleucyl-tRNA synthetase (IleRS) recognized the UAU anticodon, whereas Escherichia coli IleRS did not efficiently aminoacylate tRNA(Ile2)(UAU). In M. mobile IleRS, a single Arg residue at position 865 was critical for specificity for the UAU anticodon and, when the corresponding site (W905) in E. coli IleRS was substituted with Arg, the W905R mutant efficiently aminoacylated tRNA with UAU anticodon. Mycoplasma mobile tRNA(Ile2) cannot distinguish between AUA and AUG codon on E. coli ribosome. However, on M. mobile ribosome, M. mobile tRNA(Ile2)(UAU) specifically recognized AUA codon, and not AUG codon, suggesting M. mobile ribosome has a property that prevents misreading of AUG codon. These findings provide an insight into the evolutionary reorganization of the AUA decoding system.
Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences.
Glucocorticoids modulate diverse aspects of physiology and behavior, including energy homeostasis, stress response, and memory, through activation of the glucocorticoid receptor (GR). Light perception has profound effects on the production of glucocorticoids via functional connections of the retina to the hypothalamus-pituitary-adrenal axis. We report here that glucocorticoids can also signal in the reverse direction, i. e., regulate visual function in zebrafish, Danio rerio. The zebrafish GR mutant, gr (s357) , harbors a missense mutation that completely blocks the transcriptional activity of GR. In this mutant, visual behavior was abolished following a period of darkness and recovered sluggishly after return to the light. Electrophysiological measurements showed that the photoresponse of the dark-adapted retina was reduced in the mutant and re-adapted to light with a substantial delay. Several gene products, including some that are important for dopaminergic signaling, were misregulated in gr (s357) mutants. We suggest that GR controls a gene network required for visual adaptation in the zebrafish retina and potentially integrates neuroendocrine and sensory responses to environmental changes.
Prey capture in zebrafish larvae is an innate behavior which can be observed as early as 4~days postfertilization, the day when they start to swim. This simple behavior apparently involves several neural processes including visual perception, recognition, decision-making, and motor control, and, therefore, serves as a good model system to study cognitive functions underlying natural behaviors in vertebrates. Recent progresses in imaging techniques provided us with a unique opportunity to image neuronal activity in the brain of an intact fish in real-time while the fish perceives a natural prey, paramecium. By expanding this approach, it would be possible to image entire brain areas at a single-cell resolution in real-time during prey capture, and identify neuronal circuits important for cognitive functions. Further, activation or inhibition of those neuronal circuits with recently developed optogenetic tools or neurotoxins should shed light on their roles. Thus, we will be able to explore the prey capture in zebrafish larvae more thoroughly at cellular levels, which should establish a basis of understanding of the cognitive function in vertebrates.
We found that loss of integrity of the ribosome by removal of a putative ribosome maturation factor or a ribosomal protein conferred salt tolerance on Escherichia coli cells. Some protein synthesis inhibitors including kasugamycin and chloramphenicol also had a similar effect, although kasugamycin affected neither 16S rRNA maturation nor subunit association into a 70S ribosome. Thus, salt tolerance is a common feature of cells in which maturation or function of the ribosome is impaired. In these cells, premature induction of an alternative sigma factor, ?(E), by salt stress was observed. These results suggest the existence of a yet-unknown stress response pathway mediated by the bacterial ribosome.
In many tissues and organs, connexin proteins assemble between neighboring cells to form gap junctions. These gap junctions facilitate direct intercellular communication between adjoining cells, allowing for the transmission of both chemical and electrical signals. In rodents, gap junctions are found in differentiating myoblasts and are important for myogenesis. Although gap junctions were once believed to be absent from differentiated skeletal muscle in mammals, recent studies in teleosts revealed that differentiated muscle does express connexins and is electrically coupled, at least at the larval stage. These findings raised questions regarding the functional significance of gap junctions in differentiated muscle. Our analysis of gap junctions in muscle began with the isolation of a zebrafish motor mutant that displayed weak coiling at day 1 of development, a behavior known to be driven by slow-twitch muscle (slow muscle). We identified a missense mutation in the gene encoding Connexin 39.9. In situ hybridization found connexin 39.9 to be expressed by slow muscle. Paired muscle recordings uncovered that wild-type slow muscles are electrically coupled, whereas mutant slow muscles are not. The further examination of cellular activity revealed aberrant, arrhythmic touch-evoked Ca(2+) transients in mutant slow muscle and a reduction in the number of muscle fibers contracting in response to touch in mutants. These results indicate that Connexin 39.9 facilitates the spreading of neuronal inputs, which is irregular during motor development, beyond the muscle cells and that gap junctions play an essential role in the efficient recruitment of slow muscle fibers.
Measurement of the activity of neuronal ensembles is an essential step to understand how the neuronal network is organized and functioning. Electrical excitation of neurons causes calcium influx via voltage-gated calcium ion channels, which can be monitored by calcium imaging using fluorescent calcium probes. DNA-encoded calcium indicators (DECIs) such as cameleon and GCaMP have been developed to specifically label a subpopulation of neurons. However, in many cases, DECIs that had been developed and tested in vitro did not always show expected performance in vivo. It is necessary to increase its sensitivity and also to adjust its dynamic range to the physiological conditions. In our recent study, we developed an improved version of GCaMP and tested its performance in vivo using transgenic zebrafish. By combining the new GCaMP with targeted gene expression via the Gal4FF-UAS system, we successfully imaged the activity of the spinal motor circuit during spontaneous contractions of zebrafish larvae. Further we report here that heptanol, a gap junction blocker, could alter the spatiotemporal activation pattern of the motor circuit. Thus, we demonstrate that calcium imaging with GCaMP is powerful to analyze neuronal activities under normal and pharmacologically perturbed conditions.
Animal behaviors are generated by well-coordinated activation of neural circuits. In zebrafish, embryos start to show spontaneous muscle contractions at 17 to 19 h postfertilization. To visualize how motor circuits in the spinal cord are activated during this behavior, we developed GCaMP-HS (GCaMP-hyper sensitive), an improved version of the genetically encoded calcium indicator GCaMP, and created transgenic zebrafish carrying the GCaMP-HS gene downstream of the Gal4-recognition sequence, UAS (upstream activation sequence). Then we performed a gene-trap screen and identified the SAIGFF213A transgenic fish that expressed Gal4FF, a modified version of Gal4, in a subset of spinal neurons including the caudal primary (CaP) motor neurons. We conducted calcium imaging using the SAIGFF213A; UAS:GCaMP-HS double transgenic embryos during the spontaneous contractions. We demonstrated periodic and synchronized activation of a set of ipsilateral motor neurons located on the right and left trunk in accordance with actual muscle movements. The synchronized activation of contralateral motor neurons occurred alternately with a regular interval. Furthermore, a detailed analysis revealed rostral-to-caudal propagation of activation of the ipsilateral motor neuron, which is similar to but much slower than the rostrocaudal delay observed during swimming in later stages. Our study thus demonstrated coordinated activities of the motor neurons during the first behavior in a vertebrate. We propose the GCaMP technology combined with the Gal4FF-UAS system is a powerful tool to study functional neural circuits in zebrafish.
Since accurate translation from mRNA to protein is critical to survival, cells have developed translational quality control systems. Bacterial ribosomes stalled on truncated mRNA are rescued by a system involving tmRNA and SmpB referred to as trans-translation. Here, we review current understanding of the mechanism of trans-translation. Based on results obtained by using directed hydroxyl radical probing, we propose a new type of molecular mimicry during trans-translation. Besides such chemical approaches, biochemical and cryo-EM studies have revealed the structural and functional aspects of multiple stages of trans-translation. These intensive works provide a basis for studying the dynamics of tmRNA/SmpB in the ribosome.
We updated the tRNADB-CE by analyzing 939 complete and 1301 draft genomes of prokaryotes and eukaryotes, 171 complete virus genomes, 121 complete chloroplast genomes and approximately 230 million sequences obtained by metagenome analyses of 210 environmental samples. The 287?102 tRNA genes in total, and thus two times of the tRNA genes compiled previously, are compiled, in which sequence information, clover-leaf structure and results of sequence similarity and oligonucleotide-pattern search can be browsed. In order to pool collective knowledge with help from any experts in the tRNA research field, we included a column to which comments can be added on each tRNA gene. By compiling tRNAs of known prokaryotes with identical sequences, we found high phylogenetic preservation of tRNA sequences, especially at a phylum level. Furthermore, a large number of tRNAs obtained by metagenome analyses of environmental samples had sequences identical to those found in known prokaryotes. The identical sequence group, therefore, can be used as phylogenetic markers to clarify the microbial community structure of an ecosystem. The updated tRNADB-CE provided functions, with which users can obtain the phylotype-specific markers (e.g. genus-specific markers) by themselves and clarify microbial community structures of ecosystems in detail. tRNADB-CE can be accessed freely at http://trna.nagahama-i-bio.ac.jp.
We have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene trapping, enhancer trapping and the Gal4FF-UAS system. Gene trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and enhancer trap constructs contain the zebrafish hsp70l promoter and the GFP or Gal4FF gene. By performing genetic screens using these constructs, we have generated transgenic zebrafish that express GFP and Gal4FF in specific cells, tissues and organs. Gal4FF expression is visualized by creating double transgenic fish carrying a Gal4FF transgene and the GFP reporter gene placed downstream of the Gal4-recognition sequence (UAS). Further, the Gal4FF-expressing cells can be manipulated by mating with UAS effector fish. For instance, when fish expressing Gal4FF in specific neurons are crossed with the UAS:TeTxLC fish carrying the tetanus neurotoxin gene downstream of UAS, the neuronal activities are inhibited in the double transgenic fish. Thus, these transgenic fish are useful to study developmental biology and neurobiology.
CeR-2 RNA is one of the newly identified Caenorhabditis elegans noncoding RNAs (ncRNAs). The characterization of CeR-2 by RNomic studies has failed to classify it into any known ncRNA family. In this study, we examined the spatiotemporal expression patterns of CeR-2 to gain insight into its function. CeR-2 is expressed in most cells from the early embryo to adult stages. The subcellular localization of this RNA is analogous to that of fibrillarin, a major protein of the nucleolus. It was observed that knockdown of C/D small nucleolar ribonucleoproteins (snoRNPs), but not of H/ACA snoRNPs, resulted in the aberrant nucleolar localization of CeR-2 RNA. A mutant worm with a reduced amount of cellular CeR-2 RNA showed changes in its pre-rRNA processing pattern compared with that of the wild-type strain N2. These results suggest that CeR-2 RNA is a C/D snoRNA involved in the processing of rRNAs.
Trans-translation relieves a stalled translation on the bacterial ribosome by transfer-messenger RNA (tmRNA) with the help of SmpB, an essential cofactor of tmRNA. Here, we examined the role of the unstructured C-terminal tail of SmpB using an in vitro trans-translation system. It was found that truncation of the C-terminal tail or substitution of tryptophan residue at 147 in the middle of the C-terminal tail affected the activity in the early stage of trans-translation. Our investigations also revealed that the C-terminal tail is not required for the events until GTP is hydrolyzed by EF-Tu in complex with tmRNA-SmpB. A synthetic peptide corresponding to the C-terminal tail of SmpB inhibited peptidyl-transfer of alanyl-tmRNA and A-site binding of SmpB, but not GTP hydrolysis. These results suggest that the C-terminal tail has a role in the step of accommodation of alanyl-tmRNA-SmpB into the A-site. Directed hydroxyl radical probing indicated that tryptophan residue at 147 is located just downstream of the decoding center in the mRNA path when SmpB is in the A-site.
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.
RsgA is a unique GTP hydrolytic protein, in which the GTPase activity is significantly enhanced by the small ribosomal subunit. Depletion of RsgA causes slow cell growth as well as defects in the subunit assembly of the ribosome and the 16S rRNA processing, suggesting its involvement in the maturation of the small subunit. Several antibiotics bound to the decoding center of the small subunit inhibited the ribosome-dependent GTPase activity of RsgA. Our recent study using chemical modification indicates that the binding of RsgA induces conformational changes around the A site, P site, and helix 44. These results suggest that RsgA is involved in the maturation step of the decoding center of the small subunit of ribosome. Here, we also show a physiological role of RsgA under stress condition.
tmRNA has a dual function both as a tRNA and an mRNA to facilitate trans-translation, in which a ribosome can switch translation from a truncated or other problematic mRNA to the tmRNAs tag-encoding sequence. During trans-translation, tmRNA enters the ribosomal A-site without a codon-anticodon interaction, but with an SmpB which is a tmRNA binding protein. To further study the interaction of SmpB with ribosome and tmRNA, we have performed directed hydroxyl radical probing. It revealed that there are two SmpB binding sites at the A-site and P-site, and their C-terminal regions reside along the mRNA path in the 30S subunit. From these results, we propose a new type of molecular mimicry, in which both tmRNA and SmpB mimic the structures and functions of tRNA and mRNA during trans-translation, addressing how tmRNA preferentially recognizes the stalled ribosome, and what substitutes for a codon-anticodon interaction.
RsgA is a unique GTP hydrolytic protein in which GTPase activity is significantly enhanced by the small ribosomal subunit. Deletion of RsgA causes slow cell growth as well as defects in subunit assembly of the ribosome and 16S rRNA processing, suggesting its involvement in maturation of the small subunit. In this study, we found that removal of RsgA or inactivation of its ribosome small subunit-dependent GTPase activity provides Escherichia coli cells with resistance to high salt stress. Salt stress suppressed the defects in subunit assembly of the ribosome and processing of 16S rRNA as well as truncation of the 3 end of 16S rRNA in RsgA-deletion cells. In contrast, salt stress transiently impaired subunit assembly of the ribosome and processing of 16S rRNA and induced 3 truncation of 16S rRNA in wild-type cells. These results suggest that the action of RsgA on the ribosome, which usually facilitates maturation of the small subunit, disturbs it under a salt stress condition. Consistently, there was a drastic but transient decrease in the intracellular amount of RsgA after salt shock. Salt shock would make the pathway of maturation of the ribosome small subunit RsgA independent.
TreP [trehalose-permease (phosphotransferase system (PTS) trehalose-specific enzyme IIBC component)] is one of the target proteins of tmRNA-mediated trans-translation in Bacillus subtilis [Fujihara et al. (2002) Detection of tmRNA-mediated trans-translation products in Bacillus subtilis. Genes Cells, 7, 343-350]. The TreP synthesis is subject to CcpA-dependent carbon catabolite repression (CCR), and the treP gene contains catabolite-responsive element (cre) sequence, a binding site of repressor protein CcpA, in the coding region. Here, we demonstrated that the tmRNA-tagging of TreP occurs depending on the gene for CcpA. In the presence of CcpA, the transcription of treP mRNA terminates at 8-9 nucleotides upstream of the 5-edge of the internal cre sequence, and translational switch to the tag-sequence occurs at the 101st amino-acid (asparagine) position from N-terminus of TreP. The results show that trans-translation reaction is involved in the tagging and degradation of the N-terminal TreP fragment produced by truncated mRNA, which is a product of transcriptional roadblock by CcpA binding to the cre sequence in the internal coding region.
We constructed a new large-scale database of tRNA genes by analyzing 534 complete genomes of prokaryotes and 394 draft genomes in WGS (Whole Genome Shotgun) division in DDBJ/EMBL/GenBank and approximately 6.2 million DNA fragment sequences obtained from metagenomic analyses. This exhaustive search for tRNA genes was performed by running three computer programs to enhance completeness and accuracy of the prediction. Discordances of assignment among three programs were found for approximately 4% of the total of tRNA gene candidates obtained from these prokaryote genomes analyzed. The discordant cases were manually checked by experts in the tRNA experimental field. In total, 144,061 tRNA genes were registered in the database tRNADB-CE, and the number of the genes was more than four times of that of the genes previously reported by the database from analyses of complete genomes with tRNAscan-SE program. The tRNADB-CE allows for browsing sequence information, cloverleaf structures and results of similarity searches among all tRNA genes. For each of the complete genomes, the number of tRNA genes for individual anticodons and the codon usage frequency in all protein genes and the positioning of individual tRNA genes in each genome can be browsed. tRNADB-CE can be accessed freely at http://trna.nagahama-i-bio.ac.jp.
Channelrhodopsin (ChR)-wide receiver (ChRWR), one of the chimeric molecule of ChR1 and ChR2, has several advantages over ChR2 such as improved expression in the plasma membrane and enhanced photocurrent with small desensitization. Here we generated transgenic zebrafish (Danio rerio) expressing ChRWR as a conjugate of EGFP under the regulation of UAS promoter (UAS:ChRWR-EGFP). When crossed with a Gal4 line, SAGFF36B, ChRWR-EGFP was selectively expressed in primary mechanosensory Rohon-Beard (RB) neurons. The direct photoactivation of RB neurons was sufficient to trigger the escape behavior. The UAS:ChRWR-EGFP line could facilitate a variety of investigations of neural networks and behaviors of zebrafish in vivo.
Trans-translation is a bacterial quality control system in protein synthesis facilitated by transfer-messenger RNA (tmRNA). Here, we describe the in vitro system using purified factors to evaluate the two steps of trans-translation: peptidyl-transfer from peptidyl-tRNA to alanyl-tmRNA and decoding of the resume codon on tmRNA.
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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.