TY - JOUR. T1 - RNA-binding protein HuR interacts with thrombomodulin 5′untranslated region and represses internal ribosome entry site-mediated translation under IL-1β treatment. AU - Yeh, Chiu Hung. AU - Hung, Liang Y.. AU - Hsu, Chin. AU - Le, Shu Y.. AU - Lee, Pin T.. AU - Liao, Wan L.. AU - Lin, Yi Tseng. AU - Chang, Wen Chang. AU - Tseng, Joseph T.. PY - 2008/9. Y1 - 2008/9. N2 - Reduction in host-activated protein C levels and resultant microvascular thrombosis highlight the important functional role of protein C anticoagulant system in the pathogenesis of sepsis and septic shock. Thrombomodulin (TM) is a critical factor to activate protein C in mediating the anticoagulation and anti-inflammation effects. However, TM protein content is decreased in inflammation and sepsis, and the mechanism is still not well defined. In this report, we identified that the TM 5′ untranslated region (UTR) bearing the internal ribosome entry site (IRES) element controls TM protein expression. Using RNA ...
The large subunit of the ribosome contains the site at which peptide bonds are formed in the process of translation. Another striking feature of the large subunit is the exit tunnel. This feature begins at the site of peptide bond formation traversing 100 angstroms before opening to the cytosolic environment on the opposite side of the large subunit. It has been known for some time that the ribosome exit tunnel is the site of action for MLS antibiotics, one such example being erythromycin. More recently the exit tunnel has been shown to be involved in sensing and regulating the egress of newly synthesized peptides. As the exact mechanisms by which either macrolides such as erythromycin or nascent peptides inhibit ribosome function is not known, understanding how both of these regulatory activities are accomplished remains an important challenge in understanding ribosome structure and function. Through mutational analysis and the use of translational reporters, I have obtained results which show ...
The structure of bacterial ribosomes is composed of over 50 proteins and three large domain RNA molecules. Modifications in the rRNA require dozens of gene products but the role of these modifications in ribosome function are not fully understood or seem nonessential. It is believed that these modifications are a part of stabilizing RNA structure or RNA-protein interactions, mediate translation, or as checkpoints in ribosome assembly. The development of certain biophysical methods have helped better the understanding of how bacterial ribosome was constructed along with how its structure leads to function. Ribosome assembles improperly can lead to various diseases in human body because ribosomes assembly plays an important role in cells as RNA protein recognition. Therefore, understanding the ribosome assembly is a need to see how they connect together. Studies how ribosomes are regulated helps to figure out how and why errors occur in assembly biogenesis. The three contributions have come from ...
Ribosomes translate the genetic information contained in mRNAs into protein by linking together amino acids with the help of aminoacyl-tRNAs. In bacteria, protein synthesis stalls when the ribosome reaches the 3-end of truncated mRNA transcripts lacking a stop codon. Trans-translation is a conserved bacterial quality control process that rescues stalled ribosomes. Transfer-messenger RNA (tmRNA) and its protein partner SmpB mimic a tRNA by entering the A site of the ribosome and accepting the growing peptide chain. The ribosome releases the truncated mRNA and resumes translation on the tmRNA template. The open reading frame found on tmRNA encodes a peptide tag that marks the defective nascent peptide for proteolysis. A stop codon at the end of the open reading frame allows the ribosome to be recycled and engage in future rounds of translation.The entry of tmRNA into stalled ribosomes presents a challenge to our understanding of ribosome function because during the canonical decoding process, the
The chemical-carcinogen-induced detachment of ribosomes from rat liver endoplasmic reticulum was studied in vitro. Incubation of postmitochondrial supernatant with 0.2 mM-diethylnitrosamine or N-2-acetylaminofluorene removed approx. 16% of membrane-bound ribosomes, measured as differences in RNA/protein values of membrane separated from unbound ribosomes by flotation. These ribosomes are also detached by exposure to high centrifugal forces (160000g) and are among those removed by NADPH-catalysed lipid peroxidation. Extensive lipid peroxidation prohibits any measurement. The ribosomes (polyribosomes) removed are not those detached from the membrane by exposure to high KC1 concentrations (loosely bound) or high KC1 concentrations in the presence of puromycin (tightly bound). It is concluded then that centrifugally labile and carcinogen-sensitive represent a previously unreported sub-population of membrane-bound ribosomes. ...
Hepatitis C virus (HCV) protein synthesis is mediated by a highly conserved internal ribosome entry site (IRES), mostly located at the 5′ untranslatable region (UTR) of the viral genome. The translation mechanism is different from that used by cellular cap-mRNAs, making IRESs an attractive target site for new antiviral drugs. The present work characterizes a chimeric RNA molecule (HH363-50) composed of two inhibitors: a hammerhead ribozyme targeting position 363 of the HCV genome and an aptamer directed towards the essential stem-loop structure in domain IV of the IRES region (which contains the translation start codon). The inhibitor RNA interferes with the formation of a translationally active complex, stalling its progression at the level of 80S particle formation. This action is likely related to the effective and specific blocking of HCV IRES-dependent translation achieved in Huh-7 cells. The inhibitor HH363-50 also reduces HCV RNA levels in a subgenomic replicon system. The present findings
Ribosomes are responsible for the synthesis of all cellular proteins. It was initially believed that translating nascent chains would not interact with the ribosome exit tunnel, however, a small but increasing number of proteins have been identified that interact with the exit tunnel to induce translational arrest. Escherichia coli (E.coli) secretion monitor (SecM) is one such stalling peptide. SecM monitors the SecYEG translocon export activity through its own translocation to the periplasm and upregulates translation of SecA, an ATPase involved in the SecYEG translocation machinery, when translocation is reduced. How stalling peptides interact with the ribosome exit tunnel is not fully understood, however, a key feature required is an essential amino acid arrest motif at their C-terminus, and additionally some peptides, including SecM, undergo compaction of the nascent chain within the exit tunnel upon stalling.. In this study analysis of SecM peptides with both alanine and conservative ...
Interestingly, total RNA (i.e., ribosome density) was 2.3-fold greater in O-WD/SED versus O-WD/EX rats (p = 0.003) despite levels of upstream binding factor protein, RNA polymerase I protein and pre-45S rRNA being greater in O-WD/EX rats. Ribophagy (USP10 and G3BP1) and TRAMP-exosome rRNA degradation pathway (EXOSC10 and SKIV2L2) proteins were assayed to determine if these pathways were involved with lower ribosome density in O-WD/EX rats. While USP10 was higher in O-CON versus O-WD/SED and O-WD/EX rats (p < 0.001 and p < 0.001, respectively), G3BP1, EXOSC10 and SKIV2L2 did not differ between groups. Nop56 and Ncl mRNAs, ribosome assembly markers, were highest in O-WD/EX rats. However, Fbl mRNA and 28S rRNA, downstream ribosome processing markers, were lowest in O-WD/EX rats. Collectively these data suggest that, in WD-fed rats, endurance training increases select skeletal muscle ribosome biogenesis markers. However, endurance training may reduce muscle ribosome density by interfering with rRNA ...
Surveying the relative impact of mRNA features on local ribosome profiling read density in 28 datasets. Patrick OConnor , Dmitry Andreev , Pavel Baranov doi: http://dx.doi.org/10.1101/018762 Ribosome profiling is a promising technology for exploring gene expression. However, ribosome profiling data are characterized by a substantial number of outliers due to technical and biological factors. Here…
Ribosome biogenesis and cell cycle are coordinated processes (Du and Stillman, 2001 Bernstein and Baserga, 2004; Fatica and Tollervey, 2002; Li et al., 2009; Strezoska et al., 2002). Mutations in genes encoding factors that are involved in ribosome biogenesis cause defects in ribosomal RNA processing as well as cell cycle arrest. Recent studies with mammalian cell lines have shown that ribosome biogenesis is also linked to tumorgenesis, that is mutation or depletion of ribosomal factors, leads to cancer cell proliferation (Montanaro et al., 2008). The yeast Saccharomyces cerevisiae is a useful model organism for understanding the connections between ribosome biogenesis and cell cycle control. Only a handful of studies have been done and these have mainly focused on different transacting factors involved in ribosome biogenesis; few studies have focused on the roles of r-proteins themselves in linking cell cycle progression and rRNA processing. I wanted to investigate what roles these r-proteins ...
Related Articles Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome. Proc Natl Acad Sci U S A. 2014 May 20;111(20):7284-9 Authors: Kaushal PS, Sharma MR, Booth TM, Haque EM, Tung CS, Sanbonmatsu KY, Spremulli LL, Agrawal RK Abstract The mammalian mitochondrial ribosomes (mitoribosomes) are responsible for synthesizing 13 membrane proteins that form…
Supplement A ribosome is a molecule consisting of two subunits that fit together and work as one to build proteins according to the genetic sequence held within the messenger RNA (mRNA). Using the mRNA as a template, the ribosome traverses each codon, pairing it with the appropriate amino acid. This is done through interacting with transfer RNA (tRNA) containing a complementary anticodon on one end and the appropriate amino acid on the other. Some ribosomes occur freely in the cytosol whereas others are attached to the nuclear membrane or to the endoplasmic reticulum (ER) giving the latter a rough appearance, hence, the name rough ER or rER. Ribosomes of prokaryotes (e.g. bacteria) are smaller than most of the ribosomes of eukaryotes (e.g. plants and animals). However, the plastids and mitochondria in eukaryotes have smaller ribosomes similar to those in prokaryotes - a possible indication of the evolutionary origin of these organelles. In mid-1950s, ribosomes were first observed as dense ...
Mono- and Stereopictres of 5.0 Angstrom coordination sphere of Zinc atom in PDB 3ofq: Crystal Structure Of The E. Coli Ribosome Bound to Erythromycin. This File Contains The 50S Subunit of the Second 70S Ribosome.
Mono- and Stereopictres of 5.0 Angstrom coordination sphere of Zinc atom in PDB 3ofd: Crystal Structure Of The E. Coli Ribosome Bound to Chloramphenicol. This File Contains The 50S Subunit of the Second 70S Ribosome.
cansSAR 3D Structure of 3OFP_P | CRYSTAL STRUCTURE OF THE E. COLI RIBOSOME BOUND TO ERYTHROMYCIN. THIS FILE CONTAINS THE 30S SUBUNIT OF THE SECOND 70S RIBOSOME. | 3OFP
Helicity of membrane proteins can be manifested inside the ribosome tunnel, but the determinants of compact structure formation inside the tunnel are largely unexplored. Using an extended nascent peptide as a molecular tape measure of the ribosomal tunnel, we have previously demonstrated helix forma …
The ribosomal polypeptide tunnel exit is the site where a variety of factors interact with newly synthesized proteins to guide them through the early steps of their biogenesis. In mitochondrial ribosomes, this site has been considerably modified in the course of evolution. In contrast to all other translation systems, mitochondrial ribosomes are responsible for the synthesis of only a few hydrophobic membrane proteins that are essential subunits of the mitochondrial respiratory chain. Membrane insertion of these proteins occurs co-translationally and is connected to a sophisticated assembly process that not only includes the assembly of the different subunits but also the acquisition of redox co-factors. Here, we describe how mitochondrial translation is organized in the context of respiratory chain assembly and speculate how alteration of the ribosomal tunnel exit might allow the establishment of a subset of specialized ribosomes that individually organize the early steps in the biogenesis of distinct
The Hsp70 Ssb, which is associated with ribosomes regardless of their translational state, is the major ribosome-associated chaperone. It can be crosslinked to short nascent chains that extend only a few amino acids beyond the ribosome exit site. Like all Hsp70s, Ssb has a J-type protein as a co-chaperone. Zuo1, Ssbs J-partner binds directly to the ribosome. Surprisingly, Zuo1 forms a stable complex with an Hsp70-related protein Ssz1, tethering it to the ribosome. Ssz1 does not appear to act as a classical Hsp70 however, as deletion of the putative peptide-binding domain has no obvious effect on its in vivo function. Intriguingly, when not bound to ribosomes, Ssz1 or Zuo1 activates a signal transduction pathway. ...
Peptide-bond formation is the enzymatic activity of the ribosome. The catalytic site is made up of ribosomal RNA, indicating that the ribosome is a ribozyme. This review summarizes the recent progress in understanding the mechanism of peptide bond formation. The results of biochemical and kinetic experiments, mutagenesis studies and ribosome crystallography suggest that the approx. 107-fold rate enhancement of peptide bond formation by the ribosome is mainly due to substrate positioning within the active site, rather than to chemical catalysis.. ...
Synthetic biology technology could lead to new antibiotics, modified protein-generators. Synthetic biology researchers at Northwestern University, working with partners at Harvard Medical School, have for the first time synthesized ribosomes -- cell structures responsible for generating all proteins and enzymes in our bodies -- from scratch in a test tube.. Others have previously tried to synthesize ribosomes from their constituent parts, but the efforts have yielded poorly functional ribosomes under conditions that do not replicate the environment of a living cell. In addition, attempts to combine ribosome synthesis and assembly in a single process have failed for decades.. Michael C. Jewett, a synthetic biologist at Northwestern, George M. Church, a geneticist at Harvard Medical School, and colleagues recently took another approach: they mimicked the natural synthesis of a ribosome, allowing natural enzymes of a cell to help facilitate the man-made construction.. The technology could lead to ...
MOTIVATION: Deep sequencing based ribosome footprint profiling can provide novel insights into the regulatory mechanisms of protein translation. However, the observed ribosome profile is fundamentally confounded by transcriptional activity. In order to decipher principles of translation regulation, tools that can reliably detect changes in translation efficiency in case-control studies are needed. RESULTS: We present a statistical framework and an analysis tool, RiboDiff, to detect genes with changes in translation efficiency across experimental treatments. RiboDiff uses generalized linear models to estimate the over-dispersion of RNA-Seq and ribosome profiling measurements separately, and performs a statistical test for differential translation efficiency using both mRNA abundance and ribosome occupancy ...
Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coil HflX, a GTPase with unknown function. Our data reveal that HflX is a heat shock-induced ribosome-splitting factor capable of dissociating vacant as well as mRNA-associated ribosomes with deacylated tRNA in the peptidyl site. Structural data demonstrate that the N-terminal effector domain of HflX binds to the peptidyl transferase center in a strikingly similar manner as that of the class I release factors and induces dramatic conformational changes in central intersubunit bridges, thus promoting subunit dissociation. Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat shock, These results suggest a primary role of HflX in rescuing translationally arrested ribosomes under stress conditions.. ...
Bacterial mRNAs are translated by closely spaced ribosomes and degraded from the 5-end, with half-lives of around 2 min at 37 °C in most cases. Ribosome-free or "naked" mRNA is known to be readily degraded, but the initial event that inactivates the mRNA functionally has not been fully described. Here, we characterize a determinant of the functional stability of an mRNA, which is located in the early coding region. Using literature values for the mRNA half-lives of variant lacZ mRNAs in Escherichia coli, we modeled how the ribosome spacing is affected by the translation rate of the individual codons. When comparing the ribosome spacing at various segments of the mRNA to its functional half-life, we found a clear correlation between the functional mRNA half-life and the ribosome spacing in the mRNA region approximately between codon 20 and codon 45. From this finding, we predicted that inserts of slowly translated codons before codon 20 or after codon 45 should shorten or prolong, respectively, ...
Ribosome peptide exit tunnel plays a crucial role in the functioning of ribosomes across all domains of life.1 2 3 Before the transition of nascent peptides to mature functional proteins, they must travel through the functionally conserved peptide exit tunnel. 4 Additionally, the latent chaperone activity of the exit tunnel 5 6 suggests its role in ribosomal evolution, in the transition from short non-structured peptides to extant globular proteins. The wall of the tunnel is constructed mostly from RNA. As high as 80% of the tunnel is RNA in some species. 4 Our objective is to gain an understanding of the molecular basis of the latent chaperone activity and the preferential construction of the ribosome exit tunnel from the RNA component of the ribosome. Toward this end we have designed ketolide-peptide compounds (peptolides) to probe the mechanisms employed by the ribosome to, (i) facilitate in-tunnel folding of nascent peptides and (ii) distinguish between some peptide sequences while ...
Exhibit A: kasugamycin, an antibiotic that inhibits translation initiation in bacteria by interfering with binding of the the initiator tRNA. Amazingly enough, treatment with kasugamycin results in dramatic change in the ribosomal composition which is in turn changing ribosomes functional properties. Several proteins dissociate from the small ribosomal subunit (S1, S2, S6, S12, S18 and S21) which turns the 70S ribosome into a 61S kasugamycin particle. Ribosomal protein S1 is of particular interest here, because it is very important for the mRNA:ribosome interactions and is responsible for A/U rich sequences acting as translational activators ...
Alternative approach is to have many different ribosomes for different mRNAs. This is seemingly what we have in yeast (see above). Specific localization of different ribosomes and use of different mRNA-specific factors would then ensure proper coupling of appropriate ribosome with the right mRNA. Different localization of different paralogues of r-proteins in Saccharomyces cerevisiae is shown experimentally, and these proteins have different requirements for assembly into the 80S ...
Transfer RNA (tRNA) molecules play a crucial role in protein biosynthesis in all organisms. Their interactions with ribosomes mediate the translation of genetic messages into polypeptides. Three tRNAs bound to the Escherichia coli 70S ribosome were visualized directly with cryoelectron microscopy and three-dimensional reconstruction. The detailed arrangement of A- and P-site tRNAs inferred from this study allows localization of the sites for anticodon interaction and peptide bond formation on the ribosome. ...
Ribosomes can recognize features of a nascent peptide and exploit these features to regulate gene expression. Luis R. Cruz-Vera and Charles Yanofsky of Stanford University, Stanford, Calif., show that a bacterial nascent leader peptide, TnaC, created a binding site for tryptophan within the translating ribosome. When bound, tryptophan inhibited release factor action and stalled the ribosome. The stalled ribosome, in turn, inhibited Rho, a transcription termination factor, allowing transcription of genes of the tna operon that are essential for tryptophan catabolism.
GPR41 is a G protein-coupled receptor activated by short chain fatty acids. The gene encoding GPR41 is located immediately downstream of a related gene encoding GPR40, a receptor for long chain fatty acids. Expression of GPR41 has been reported in a small number of cell types, including gut enteroendocrine cells and sympathetic ganglia, where it may play a role in the maintenance of metabolic homeostasis. We now demonstrate that GPR41, like GPR40, is expressed in pancreatic beta cells. Surprisingly, we found no evidence for transcriptional control elements or transcriptional initiation in the intergenic GPR40-GPR41 region. Rather, using 5-rapid amplification of cDNA ends analysis, we demonstrated that GPR41 is transcribed from the promoter of the GPR40 gene. We confirmed this finding by generating bicistronic luciferase reporter plasmids, and we were able to map a potential internal ribosome entry site-containing region to a 2474-nucleotide region of the intergenic sequence. Consistent with this, we
Ribosomes are comprised of 65% RNA and 35% proteins. Ribosomes are cellular organelles that are responsible for Protein Synthesis. Ribosomes function
The determination of the high-resolution structures of ribosomal subunits in the year 2000 and of the entire ribosome a few years later are revolutionizing our understanding of the role of the ribosome in translation. In the present article, I summarize the main contributions from our laboratory to this worldwide effort. These include the determination of the structure of the 30S ribosomal subunit and its complexes with antibiotics, the role of the 30S subunit in decoding, and the high-resolution structure of the entire 70S ribosome complexed with mRNA and tRNA.. ...
Distance vectors from the center of mass of each identified polysomal ribosome to the center of mass of its closest neighbor particle clearly indicated preferential positioning at a distance around 22±2 nm. Researchers analysis reveals a remarkably well defined arrangement of ribosomes in staggered or helical orientations, with the mRNA being sequestered on the inside, while the tRNA entrance sites and the polypeptide exit sites are exposed to the cytosol. See BNID 105001. Note-Martin and Miller 1983, PMID 6683685 p. 345 right column 2nd paragraph write that a ribosome binds every ~135 nts in sea urchin ...
Affinity proteins are invaluable tools in biotechnological and medical applications. This thesis is about combinatorial protein engineering principles for the generation of novel affinity proteins to purify mouse immunoglobulin, detect a potential cancer marker protein or inhibit a cell proliferation pathway.. In a first study, ribosome display was for the first time applied to the selection of so-called affibody molecules, including the design of a ribosome display gene cassette, initial test enrichment experiments and the selection of binders against murine IgG1. One of the selected binders (ZMAB25) showed a highly selective binding profile to murine IgG1, which was exploited in the recovery of two different mouse monoclonal IgG1 antibodies from a bovine immunoglobulin-containing background. Ribosome display was further applied to the selection of affibody molecules binding to SATB1, a suggested marker protein for metastasizing adenocarcinoma. The study also included the selection of VHH ...
Ribosomes synthesizing nascent secretory proteins are targeted to the membrane by the signal recognition particle (SRP), a small ribonucleoprotein that binds to the signal peptide as it emerges from the ribosome. SRP arrests further elongation, causing ribosomes to stack behind the arrested ribosome. Upon interaction of SRP with its receptor on the ER membrane, the translation arrest is released and the ribosome becomes bound to the ER membrane. We have examined the distribution of unattached and membrane-bound ribosomes during the translation of mRNAs encoding two secretory proteins, bovine preprolactin and rat preproinsulin I. We find that the enhancement of ribosome stacking that occurs when SRP arrests translation of these proteins is relaxed in the presence of microsomal membranes. We also demonstrate that two previously described populations of membrane-associated ribosomes, distinguished by their sensitivity to high salt or EDTA extraction, correspond to ribosomes that have synthesized ...
The nucleus is where the DNA is kept and RNA is transcribed. RNA is moved out of the nucleus through the nuclear pores. Proteins needed inside the nucleus are transported in through the nuclear pores. The nucleolus is usually visible as a dark spot in the nucleus, and is the location of ribosome formation. Ribosomes are where RNA is translated into protein. This process is called protein synthesis. Protein synthesis is very important to cells, therefore large numbers of ribosomes are found in cells. Ribosomes float freely in the cytoplasm, and are also bound to the endoplasmic reticulum (ER). ER bound to ribosomes is called rough ER because the ribosomes on the ER give it a rough sandpaper like look.. These organelles are very small, made up of 50 proteins and several long RNAs bound together. Ribosomes do not have a membrane. Ribosomes fall into two seperate units while not synthesizing protein.. The endoplasmic reticulum is the transport system for molecules needed for certain changes and ...
The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent
We propose that the lysis gene of RNA phage GA is translated exclusively by ribosomes coming from the upstream coat gene on the basis of the following observations. (i) The initiation codon AUG of the lysis gene is well hidden by the possible hairpin structure. (ii) There is no functional SD or SD‐like sequence near the initiation codon AUG. (iii) Elimination of the upstream sequence completely abolished the expression of the downstream lysis gene. Furthermore, introduction of the SD sequence into this construct restored lysis gene expression even without the upstream coat gene. (iv) RRF may not release ribosomes from the unusual gene border sequence UAAUG. (v) The ribosomes translating the upstream coat gene must come into close proximity with the initiation codon of the downstream lysis gene either by a nucleotide distance of not more than two nucleotides or by RNA secondary structure.. Having established that the lysis gene is translated exclusively by the upstream ribosomes, this system is ...
Ribosomes are large ribonucleoprotein complexes which incorporate amino acids into peptide chains during translational process in all types of living cells. The eukaryotic ribosome is larger compared to its prokaryotic counterpart. The size differences are due to a larger protein part and that the rRNA contains eukaryote specific expansion segments (ES). Cryo-EM reconstruction has visualized many ES on the ribosomal surface which have given clues about function and structural features. However, the secondary structures of most ES are unknown or ill defined. In this thesis, the secondary and also to a certain extent the tertiary structures of several ES are determined by using computational methods and biochemical experimental techniques. The juxtaposition of ES6 close to ES3 in the Cryo-EM image of the yeast ribosome suggested that ES3 and ES6 might interact. A computational analysis of more than 2900 sequences shows that a complementary helical region of seven to nine contiguous base pairs can ...
The ribosome is a molecular witness to the origin of life as it comprises the conserved mechanism for the translation of nucleic acid gene sequences into proteins in all living creatures. The large subunit is reducible to a core peptidyl-transferase structure, which is proposed as its ancestral form. A model of hierarchical addition of E. coli 23S rRNA modular inserts was recently proposed to explain ribosomal evolution from this core structure, however large numbers of alternative chronologies are possible. Our refinement of this model using the Design Structure Matrix (DSM) provides a surprisingly detailed picture of the transitional events. Similar methods can be adapted to the chronology model of ribosome small subunit of E. coli, with which, the evolutionary story of the integrated E. coli ribosome can be presented as a 3D animation using Autodesk Maya. It would enlarge our knowledge about the RNA world approximately 3.8 billion years ago ...
Ribosomes are giant molecular machines that produce all proteins necessary for life. In eukaryotic cells, their assembly is a highly elaborate and carefully coordinated process. The Klinge labs research is aimed at understanding the molecular mechanisms that govern early stages of eukaryotic ribosome assembly. Ribosomes are responsible for decoding the information contained in messenger RNA to synthesize proteins used in all domains of life. Ribosome assembly, the process by which ribosomes are synthesized, involves approximately 200 protein and RNA factors in eukaryotes, most of which are essential. These factors are involved in all stages of ribosome assembly, from transcription of ribosomal RNA in the nucleolus to export into the cytoplasm, where the final stages of maturation and quality control occur. As ribosome assembly progresses, more and more of this machinery is released from intermediate complexes until the ribosomal subunits complete maturation.. The structure of this molecular ...
Protein synthesis is a complex process performed by sophisticated cellular particles known as ribosomes. Although RNA constitutes the major structural and functional component, ribosomes from all kingdoms contain an extensive array of proteins with largely undefined functional roles. The work presented in this thesis addresses ribosomal complexity using mutants of Salmonella typhimurium to examine the physiological effects of ribosomal protein (r-protein) removal and orthologous replacement on bacterial fitness and ribosome function.. The results of paper I demonstrate that removal of small subunit protein S20 conferred two independent translation initiation defects: (i) a significant reduction in the rate and extent of mRNA binding and (ii) a drastic decrease in the yield of 70S complexes caused by an impairment in subunit association. The topographical location of S20 in mature 30S subunits suggests that these perturbations are the result of improper orientation of helix 44 of the 16S rRNA ...
Definition of ribosome inactivating proteins in the Definitions.net dictionary. Meaning of ribosome inactivating proteins. What does ribosome inactivating proteins mean? Information and translations of ribosome inactivating proteins in the most comprehensive dictionary definitions resource on the web.
Our research focuses on how ribosomal subunits are assembled in eukaryotic cells. We use the yeast S. cerevisiae as a model organism to study this essential and highly conserved process. Ribosomes are among the largest and most complex macromolecular machines assembled in cells. Ribosomes are assembled from imported ribosomal proteins on nascent rRNA in the nucleolus of the cell. The large and small subunits are then independently exported through nuclear pores at rates that can reach 30 subunits per second. Both subunits require the export receptor Crm1 for export, and both undergo independent post-export maturation events that are required before the subunits are translation-ready. Over 150 proteins that do not end up as constituents of the mature ribosome are necessary for ribosome biogenesis. The molecular details of how most of these non-ribosomal proteins function is unknown. We have focused our work on the late steps of small subunit biogenesis. In particular, we are characterizing the ...
Ribosomes, the molecular machines responsible for assembling protein from amino acids, are among the most fundamental structures in biology and are found across all domains of life. These machines are assembled hierarchically by the association of 54 proteins to three strands of RNA (in bacteria) in a highly coordinated sequence. My research involves the construction of a lattice based, computational model of Escherichia coli to study the in vivo assembly of the ribosomal small subunit. Explicit in this model is the transcription and translation of the ribosomal components. The central cell in this figure is a visualization of a single time point in the simulation. The cubes show how the simulation domain is discretized onto a lattice. The spheres represent the ribosomal protein (gray), small (yellow) and large (brown) subunits, completed ribosomes (white), and assembling ribosomes (orange). Inside the cell is a representation of the bacterial chromosome from which the ribosomal constituents are ...
Simian virus 40-based plasmids that direct the synthesis of preproinsulin during short-term transfection of COS cells have been used to probe the mechanism of reinitiation by eucaryotic ribosomes. Earlier studies from several laboratories had established that the ability of ribosomes to reinitiate translation at an internal AUG codon depends on having a terminator codon in frame with the preceding AUG triplet and upstream from the intended restart site. In the present studies, the position of the upstream terminator codon relative to the preproinsulin restart site has been systematically varied. The efficiency of reinitiation progressively improved as the intercistronic sequence was lengthened. When the upstream "minicistron" terminated 79 nucleotides before the preproinsulin start site, the synthesis of proinsulin was as efficient as if there were no upstream AUG codons. A mechanism is postulated that might account for this result, which is somewhat surprising inasmuch as bacterial ribosomes ...
A sequence-specific modification of the human 5.8 S rRNA in isolated 60 S subunits, non-programmed 80 S ribosomes and ribosomes complexed with mRNA and tRNAs was studied with the use of a derivative of the nonaribonucleotide UCUGUGUUU bearing a perfluorophenylazide group on the C-5 atom of the 5′-terminal uridine. Part of the oligonucleotide moiety of the derivative was complementary to the 5.8 S rRNA sequence ACACA in positions 82-86 flanked by two guanines at the 5′-terminus. The target for the cross-linking was identified as nucleotide G89 on the 5.8 S RNA. In addition, several ribosomal proteins were modified by the oligonucleotide derivative bound to the 5.8 S rRNA and proteins L6 and L8 were among them. Application of these results to known cryo-electron microscopy images of eukaryotic 60 S subunits made it possible to suggest that the central part of the 5.8 S rRNA containing the sequence 82-86 and proteins L6 and L8 are located at the base of the L1 stalk of the 60 S subunit. The ...
Cambio, distributor of molecular biology reagents and consumables to scientific research laboratories within the UK, has added an innovative new product to its portfolio. ARTseq™ (Active mRNA Translation) ribosome profiling kits, produced by Epicentre, enable users to create RNA sequence libraries from ribosome-protected mRNA. This novel technique is used to investigate translational control, measure gene expression, identify translation start sites and predict protein abundance, making them ide
The ribosome uses this mRNA as template and translates each codon by pairing it with appropriate amino acid which is provided by aminoacyl-tRNA (aminoacyl-tRNA contains a complementary anti-codon). The ribosome contains three RNA binding sites as A, P and E. The A site binds an aminoacyl-tRNA; the P site binds a peptidyl-tRNA and E site binds a free tRNA before it exits the ribosome as can be seen in the diagram ...
Ribosome stalling during translation has recently been shown to cause neurodegeneration, yet the signaling pathways triggered by stalled elongation complexes are unknown. To investigate these pathways we analyzed the brain of C57BL/6J-Gtpbp2(nmf205)(-/-) mice in which neuronal elongation complexes are stalled at AGA codons due to deficiencies in a tRNA(Arg)UCU tRNA and GTPBP2, a mammalian ribosome rescue factor. Increased levels of phosphorylation of eIF2α (Ser51) were detected prior to neurodegeneration in these mice and transcriptome analysis demonstrated activation of ATF4, a key transcription factor in the integrated stress response (ISR) pathway. Genetic experiments showed that this pathway was activated by the eIF2α kinase, GCN2, in an apparent deacylated tRNA-independent fashion. Further we found that the ISR attenuates neurodegeneration in C57BL/6J-Gtpbp2(nmf205)(-/-) mice, underscoring the importance of cellular and stress context on the outcome of activation of this pathway. These results
Camara Y., Asin-Cayuela J., Park C.B., Metodiev M.D., Shi Y., Ruzzenente B., Kukat C., Habermann B., Wibom R., Hultenby K., Franz T., Erdjument-Bromage H., Tempst P., Hallberg B.M., Gustafsson C.M., Larsson N.G.. Precise control of mitochondrial DNA gene expression is critical for regulation of oxidative phosphorylation capacity in mammals. The MTERF protein family plays a key role in this process, and its members have been implicated in regulation of transcription initiation and site-specific transcription termination. We now demonstrate that a member of this family, MTERF4, directly controls mitochondrial ribosomal biogenesis and translation. MTERF4 forms a stoichiometric complex with the ribosomal RNA methyltransferase NSUN4 and is necessary for recruitment of this factor to the large ribosomal subunit. Loss of MTERF4 leads to defective ribosomal assembly and a drastic reduction in translation. Our results thus show that MTERF4 is an important regulator of translation in mammalian ...