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At least 55 records · Page 3Linked to original sources
PubMed1989

Unusual mRNA pseudoknot structure is recognized by a protein translational repressor.

Translation of ribosomal proteins in the alpha operon of E. coli is repressed by one of the encoded proteins, S4; it specifically recognizes an RNA fragment containing the translational initiation site for the first gene in the operon. RNA structure mapping experiments have suggested a pseudoknot structure for the S4 binding site: the loop of a hairpin is base paired to sequences downstream of the hairpin. Here, we systematically test this proposed structure by measuring S4 binding to an extensive set of site-directed mutations that create compensatory base pair changes in potential helices. The pseudoknot folding is confirmed, and two additional, unexpected interactions within the pseudoknot are also detected. The overall structure is an unusual "double pseudoknot" linking a hairpin upstream of the ribosome binding site with sequences 2-10 codons downstream of the initiation codon. Stabilization of this structure by S4 could account for translational repression.

Base Composition↗
PubMed1991

Synthesis and developmental regulation of an egg specific mouse protein translated from maternal mRNA.

Proteins synthesized by DDK mice embryos were analyzed by 2D electrophoresis and a new egg-specific polypeptide, D14, was identified. The protein is characterized by its high rate of synthesis and electrophoretic properties (MW 36,500, pl greater than 8). The synthesis of D14 is strictly developmentally regulated: starting in the maturing oocyte in the few hours following germinal vesicle breakdown (GVBD), it remains high over the first cell cycle and decreases abruptly during the two-cell stage. The arrest of D14 synthesis is triggered by egg activation and does not directly depend on transcription by the zygotic genome. Nevertheless, drugs that perturb the onset of zygotic transcription concomitantly inhibit D14 arrest of synthesis. D14 is present in both cytoplasmic and nuclear compartments at the two-cell stage; it is very stable and remains detectable at least until the eight-cell stage in the preimplantation embryo. Embryos of wild strains of mice synthesized either D14 or a D14 related polypeptide at a rate comparable to that of DDK embryos, which was at least ten times greater than that found in other laboratory strains. Both the developmental regulation and the genetic variability in its rate of synthesis make D14 an interesting polypeptide for the study of regulation of maternal information in the very early stages of mouse embryo development.

Animals↗
PubMed1999

Restricted virus protein translation in canine distemper virus inclusion body polioencephalitis.

In this study, inclusion body polioencephalitis, an uncommon form of canine distemper virus (CDV)-induced encephalitis, was investigated for viral protein and mRNA expression by immunohistochemistry (IH) and in situ hybridization and, in addition, infiltrating cells were characterized by IH. Lesions were predominantly found in the grey matter of the brain stem and the immune response, dominated by T cells, was associated with a strong MHC II upregulation. Abundant expression of all viral protein mRNAs and reduced or lacking protein translation, especially of the matrix protein were the most important findings, indicating that restricted virus infection in the grey matter might represent a mechanism for viral persistence in distemper polioencephalitis.

Animals↗
PubMed2006

Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL.

In a newly isolated temperature-sensitive lethal Escherichia coli mutant affecting the chaperonin GroEL, we observed wholesale aggregation of newly translated proteins. After temperature shift, transcription, translation, and growth slowed over two to three generations, accompanied by filamentation and accretion (in approximately 2% of cells) of paracrystalline arrays containing mutant chaperonin complex. A biochemically isolated inclusion body fraction contained the collective of abundant proteins of the bacterial cytoplasm as determined by SDS/PAGE and proteolysis/MS analyses. Pulse-chase experiments revealed that newly made proteins, but not preexistent ones, were recruited to this insoluble fraction. Although aggregation of "stringent" GroEL/GroES-dependent substrates may secondarily produce an "avalanche" of aggregation, the observations raise the possibility, supported by in vitro refolding experiments, that the widespread aggregation reflects that GroEL function supports the proper folding of a majority of newly translated polypeptides, not just the limited number indicated by interaction studies and in vitro experiments.

Chaperonin 60↗
PubMed2005

A model of protein translation including codon bias, nonsense errors, and ribosome recycling.

We present and analyse a model of protein translation at the scale of an individual messenger RNA (mRNA) transcript. The model we develop is unique in that it incorporates the phenomena of ribosome recycling and nonsense errors. The model conceptualizes translation as a probabilistic wave of ribosome occupancy traveling down a heterogeneous medium, the mRNA transcript. Our results show that the heterogeneity of the codon translation rates along the mRNA results in short-scale spikes and dips in the wave. Nonsense errors attenuate this wave on a longer scale while ribosome recycling reinforces it. We find that the combination of nonsense errors and codon usage bias can have a large effect on the probability that a ribosome will completely translate a transcript. We also elucidate how these forces interact with ribosome recycling to determine the overall translation rate of an mRNA transcript. We derive a simple cost function for nonsense errors using our model and apply this function to the yeast (Saccharomyces cervisiae) genome. Using this function we are able to detect position dependent selection on codon bias which correlates with gene expression levels as predicted a priori. These results indirectly validate our underlying model assumptions and confirm that nonsense errors can play an important role in shaping codon usage bias.

Animals↗
PubMed1990

Effect of the preS1 RNA sequence on the efficiency of the hepatitis B virus preS2 and S protein translation.

The gene coding for hepatitis B virus surface antigen consists of preS1, preS2, and S regions. Two species of mRNAs of this gene are transcribed. The larger species covers all three regions and is translated solely into preS1 protein, whereas the smaller one covers the preS2 and S regions and is translated into preS2 and S proteins. This study examines the influence of the 5' upstream sequence lying in the preS1 region on the synthesis of preS2 and S proteins. For this purpose, several expression plasmids were constructed by inserting various portions of the preS1 region between the retroviral LTR promoter and the preS2/S coding region, and preS2/S protein production was examined in the transfected CHL cells. All the transcripts were initiated in the LTR. A sequence located in the region between 102 and 38 nucleotides upstream from the preS2 initiation codon was found to reduce the production of preS2/S proteins probably at the level of translation. Expression of the heterologous chloramphenicol acetyltransferase gene was similarly inhibited when it was placed downstream of the preS1-102/-38 sequence.

Animals↗
PubMed2001

Ratcheting in post-translational protein translocation: a mathematical model.

We have developed a non-steady-state mathematical model describing post-translational protein translocation across the endoplasmic reticulum membrane. Movement of the polypeptide chain through the channel in the endoplasmic reticulum membrane is considered to be a stochastic process which is biased at the lumenal side of the channel by the binding of BiP (Kar2p), a member of the Hsp70 family of ATPases (ratcheting model). Assuming that movement of the chain through the channel is caused by passive diffusion (Brownian ratchet), the model describes all available experimental data. The optimum set of model parameters indicates that the ratcheting mechanism functions at near-maximum rate, being relatively insensitive to variations of the association or dissociation rate constants of BiP or its concentration. The estimated rate constant for diffusion of a polypeptide inside the channel indicates that the chain makes contact with the walls of the channel. Since fitting of the model to the data required that the backward rate constant be larger than the forward constant during early diffusion steps, translocation must occur against a force. The latter may arise, for example, from the unfolding of the polypeptide chain in the cytosol. Our results indicate that the ratchet can transport polypeptides against a free energy of about 25 kJ/mol without significant retardation of translocation. The modeling also suggests that the BiP ratchet is optimized, allowing fast translocation to be coupled with minimum consumption of ATP and rapid dissociation of BiP in the lumen of the ER. Finally, we have estimated the maximum hydrophobicity of a polypeptide segment up to which lateral partitioning from the channel into the lipid phase does not result in significant retardation of translocation.

Adenosine Triphosphate↗
PubMed1996

Intracellular cleavage of hepatitis C virus RNA and inhibition of viral protein translation by hammerhead ribozymes.

To determine the effects of hammerhead ribozymes against hepatitis C virus (HCV) RNA on viral protein translation, a luciferase reporter gene vector, pCMV/T7-NCRCdelta-luc, was constructed containing the 5'-noncoding region (5'-NCR) and part of the core region of HCV. Four ribozymes, Rz1-Rz4, were designed to cleave at nucleotide positions 136-160, 313-337, 496-520, and 373-388, respectively. Each ribozyme cleaved the target RNA at expected positions under cell-free conditions. Rz2 and Rz4 significantly suppressed translation of NCRCdelta-luc RNA by 71 and 49%, respectively. Translation of control luciferase mRNA lacking viral elements was not affected by the ribozymes. Furthermore, when NCRCdelta-luc RNA and ribozymes were cotransfected into cells, Rz2 and Rz4 significantly suppressed expression by 73 and 56%, respectively. In contrast, cleavage-deficient ribozymes with a point mutation in the hammerhead domain had no significant effect. To determine the effects of endogenously produced ribozymes, eukaryotic expression vectors for Rz2 and Rz4 were constructed. Cotransfection of the vectors with CMV/T7-NCRCdelta-luc showed suppression of luciferase activities to 50 and 61%, respectively. Moreover, transfection of pCMV/T7-NCRCdelta-luc into stable Rz2 and Rz4 producer cells also showed substantial inhibition of luciferase activity. Ribozymes directed against the HCV genome can substantially and specifically inhibit viral gene expression under intracellular conditions.

Cells, Cultured↗
PubMed1996

Post-translational protein translocation: not all hsc70s are created equal.

Hsc70s in the yeast endoplasmic reticulum (ER) and mitochondria interact with membrane-associated components of the translocation machinery and are required for post-translational protein import. Although it has been proposed that the mitochondrial and ER machines function similarly, a variety of experiments suggest that BiP, the ER hsc70, might play a more elaborate role.

Carrier Proteins↗
PubMed2026

Manipulation of protein translation and stem cell self-renewal by CRISPR activation of rRNA transcription.

Ribosomal RNA (rRNA) transcription rates vary during development, and their dysregulation is linked to diseases such as cancer and ribosomopathies. Owing to their high abundance and genomic redundancy, the functional significance of rRNA levels remains unclear. We developed TAPIR (Targeted Activation of Protein Translation), a CRISPR-based approach to elevate rRNA levels by inducing 47S ribosomal DNA transcription. TAPIR increased nucleolar size and enhanced protein synthesis, even in rapidly proliferating cells. In neural stem cells, elevated translation promoted self-renewal and proliferation in vitro and in vivo. Furthermore, TAPIR enabled the modeling and partial rescue of associated disease phenotypes. Our findings reveal that rRNA levels directly regulate translational output and that protein synthesis capacity can act as a key determinant of mammalian stem cell behavior.

Animals↗
PubMed2002

Visibly stressed: the role of eIF2, TIA-1, and stress granules in protein translation.

Eukaryotic cells express a family of eukaryotic translation initiation factor 2 alpha (eIF2alpha) kinases (eg, PKR, PERK-PEK, GCN2, HRI) that are individually activated in response to distinct types of environmental stress. Phosphorylation of eIF2alpha by one or more of these kinases reduces the concentration of eIF2-guanosine triphosphate (GTP)-transfer ribonucleic acid for methionine (tRNA(Met)), the ternary complex that loads tRNA(Met) onto the small ribosomal subunit to initiate protein translation. When ternary complex levels are reduced, the related RNA-binding proteins TIA-1 and TIAR promote the assembly of a noncanonical preinitiation complex that lacks eIF2-GTP-tRNA(Met). The TIA proteins dynamically sort these translationally incompetent preinitiation complexes into discrete cytoplasmic domains known as stress granules (SGs). RNA-binding proteins that stabilize or destabilize messenger RNA (mRNA) are also recruited to SGs during stress. Thus, TIA-1 and TIAR act downstream of eIF2alpha phosphorylation to promote SG assembly and facilitate mRNA triage during stress. The role of the SG in the integration of translational efficiency, mRNA stability, and the stress response is discussed.

Eukaryotic Initiation Factor-2↗
PubMedDate not supplied

Coding theory based models for protein translation initiation in prokaryotic organisms.

Our research explores the feasibility of using communication theory, error control (EC) coding theory specifically, for quantitatively modeling the protein translation initiation mechanism. The messenger RNA (mRNA) of Escherichia coli K-12 is modeled as a noisy (errored), encoded signal and the ribosome as a minimum Hamming distance decoder, where the 16S ribosomal RNA (rRNA) serves as a template for generating a set of valid codewords (the codebook). We tested the E. coli based coding models on 5' untranslated leader sequences of prokaryotic organisms of varying taxonomical relation to E. coli including: Salmonella typhimurium LT2, Bacillus subtilis, and Staphylococcus aureus Mu50. The model identified regions on the 5' untranslated leader where the minimum Hamming distance values of translated mRNA sub-sequences and non-translated genomic sequences differ the most. These regions correspond to the Shine-Dalgarno domain and the non-random domain. Applying the EC coding-based models to B. subtilis, and S. aureus Mu50 yielded results similar to those for E. coli K-12. Contrary to our expectations, the behavior of S. typhimurium LT2, the more taxonomically related to E. coli, resembled that of the non-translated sequence group.

Bacterial Proteins↗
PubMed2002

Upstream AUGs modulate prion protein translation in vitro.

The level of expression of the host PrP gene (PRNP) has been shown to affect the progression to a disease, transmissible spongiform encephalopathy (TSE). In order to define sequences that are responsible for translation ofPRNPmRNA we have investigated a region comprising its 5'-leader sequence. Most remarkable, it consists of an almost identical Kozak mRNA sequence and two AUG initiation codons which seem to modulate translation of the prion protein mRNA in vitro. Although transcriptional regulation of the prion protein PRNP gene had been expected to dominate the translational modulation, our observations point to a translational regulation of the mouse prion protein synthesis controlled by ribosomal entry and usage of AUG codons.

5' Untranslated Regions↗
PubMed1998

Purification and characterization of a new eukaryotic protein translation factor. Eukaryotic initiation factor 4H.

A new protein with translational activity has been identified on the basis of its ability to stimulate translation in an in vitro globin synthesis assay deficient in eukaryotic initiation factor (eIF) 4B and eIF4F. This protein has been purified to greater than 80% homogeneity from rabbit reticulocyte lysate and has been given the name eIF4H. eIF4H was shown to stimulate the in vitro activities of eIF4B and eIF4F in globin synthesis, as well as the in vitro RNA-dependent ATPase activities of eIF4A, eIF4B, and eIF4F. Three tryptic fragments of eIF4H yielded amino acid sequences that were 100% identical to a human sequence found in the GeneBankTM that codes for a previously uncharacterized protein (HUMORFU_1). The calculated molecular weight of the protein encoded by this sequence, its predicted cyanogen bromide fragmentation, and calculated isoelectric point are all consistent with those determined experimentally for eIF4H. Also, the presence of an RNA recognition motif within HUMORFU_1 suggests that eIF4H may interact with mRNA. We conclude that this newly characterized protein, eIF4H, functions to stimulate the initiation of protein synthesis at the level of mRNA utilization, and is encoded by the gene for HUMORFU_1.

Amino Acid Sequence↗
PubMed2006

Iron metabolism in insect disease vectors: mining the Anopheles gambiae translated protein database.

All animals require iron for survival. This requirement reflects the role of this mineral as a cofactor of numerous proteins. However, under physiological conditions, Fe(2+) oxidizes to Fe(3+) encouraging the formation of toxic free radicals. In mammals, the potential for oxidative damage from iron is minimized by binding iron to proteins. Mammalian iron metabolism is complex and numerous proteins are involved in iron absorption, transport, uptake and utilization. We have analyzed the Anopheles gambiae translated protein database for candidates that show identity to proteins involved in mammalian iron metabolism (Holt et al., 2002. The genome sequence of the malaria mosquito Anopheles gambiae. Science 298, 129-149). Our results indicate that proteins involved in iron absorption and intracellular iron utilization are, for the most part, conserved in A. gambiae. In contrast, proteins involved in the pathways of iron export from the gut, transport in hemolymph and uptake at peripheral tissues in mosquitos differ from those for mammals.

Animals↗
PubMed2001

Post-translational protein modifications in antigen recognition and autoimmunity.

It is estimated that 50-90% of the proteins in the human body are post-translationally modified. In the proper context, these modifications are necessary for the biological functions of a vast array of proteins and the effector functions of the cells in which they reside. However, it is now clear that some post-translational modifications can create new self antigens (Ags) or even mask Ags normally recognized by the immune system. In either case, they profoundly affect the recognition of Ag by bone marrow-derived cells, as well as their effector functions. How do post-translational protein modifications affect the processing of foreign and self Ags and what is their role in the origin of autoimmune responses?

Animals↗
PubMed1995

Structural and functional analyses of the transcription-translation proteins NusB and NusE.

The NusB and NusE (ribosomal protein S10) proteins function in transcription and translation. The two proteins form a complex that binds to the boxA sequence found in the leader RNA of rrn operons; boxA is required for transcription antitermination in rrn operons. Although binding of these two proteins to the boxA RNA of the bacteriophage lambda nut site has not been observed, both NusB and NusE as well as the RNA boxA sequence are required for lambda N-mediated antitermination. Studies identifying the amino acid changes caused by mutations in nusB and nusE and relating these changes to altered function are reported. It is concluded that boxA is essential for an effective NusB contribution to N-mediated antitermination and that by mutation NusB may be changed to allow more-effective binding to boxA variants.

Alleles↗