Reassembling green fluorescent protein for in vitro evaluation of trans-translation.
Ontology highlight
ABSTRACT:
SUBMITTER: Guyomar C
PROVIDER: S-EPMC7038980 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:When the bacterial ribosome stalls on a truncated mRNA, transfer-messenger RNA (tmRNA) acts initially as a transfer RNA (tRNA) and then as a messenger RNA (mRNA) to rescue the ribosome and add a peptide tag to the nascent polypeptide that targets it for degradation. Ribosomal protein S1 binds tmRNA but its functional role in this process has remained elusive. In this report, we demonstrate that, in vitro, S1 is dispensable for the tRNA-like role of tmRNA but is essential for its mRNA function. Increasing or decreasing the amount of protein S1 in vivo reduces the overall amount of trans-translated proteins. Also, a truncated S1 protein impaired for ribosome binding can still trigger protein tagging, suggesting that S1 interacts with tmRNA outside the ribosome to keep it in an active state. Overall, these results demonstrate that S1 has a role in tmRNA-mediated tagging that is distinct from its role during canonical translation.
| S-EPMC1874662 | biostudies-literature
Project description:Transfer-messenger RNA (tmRNA) plays a dual role as a tRNA and an mRNA in trans-translation, during which the ribosome replaces mRNA with tmRNA encoding the tag-peptide. These processes have been suggested to involve several tmRNA-binding proteins, including SmpB and ribosomal protein S1. To investigate the molecular mechanism of trans-translation, we developed in vitro systems using purified ribosome, elongation factors, tmRNA and SmpB from Thermus thermophilus. A stalled ribosome in complex with polyphenylalanyl-tRNA(Phe) was prepared as a target of tmRNA. A peptidyl transfer reaction from polyphenylalanyl-tRNA(Phe) to alanyl-tmRNA was observed in an SmpB-dependent manner. The next peptidyl transfer to aminoacyl-tRNA occurred specifically to the putative resume codon for the tag-peptide, which was confirmed by introducing a mutation in the codon. Thus, the in vitro systems developed in this study are useful to investigate the early steps of trans-translation. Using these in vitro systems, we investigated the function of ribosomal protein S1, which has been believed to play a role in trans-translation. Although T. thermophilus S1 tightly bound to tmRNA, as in the case of Escherichia coli S1, it had little or no effect on the early steps of trans-translation.
| S-EPMC1831857 | biostudies-literature
Project description:Wild-type green fluorescent protein (GFP) folds on a time scale of minutes. The slow step in folding is a cis-trans peptide bond isomerization. The only conserved cis-peptide bond in the native GFP structure, at P89, was remodeled by the insertion of two residues, followed by iterative energy minimization and side chain design. The engineered GFP was synthesized and found to fold faster and more efficiently than its template protein, recovering 50% more of its fluorescence upon refolding. The slow phase of folding is faster and smaller in amplitude, and hysteresis in refolding has been eliminated. The elimination of a previously reported kinetically trapped state in refolding suggests that X-P89 is trans in the trapped state. A 2.55 Å resolution crystal structure revealed that the new variant contains only trans-peptide bonds, as designed. This is the first instance of a computationally remodeled fluorescent protein that folds faster and more efficiently than wild type.
| S-EPMC3970891 | biostudies-literature
Project description:While BDNF is receiving considerable attention for its role in synaptic plasticity and in nervous system dysfunction, identifying brain circuits involving BDNF-expressing neurons has been challenging. BDNF levels are very low in most brain areas, except for the large mossy fiber terminals in the hippocampus where BDNF accumulates at readily detectable levels. This report describes the generation of a mouse line allowing the detection of single brain cells synthesizing BDNF. A bicistronic construct encoding BDNF tagged with a P2A sequence preceding GFP allows the translation of BDNF and GFP as separate proteins. Following its validation with transfected cells, this construct was used to replace the endogenous Bdnf gene. Viable and fertile homozygote animals were generated, with the GFP signal marking neuronal cell bodies translating the Bdnf mRNA. Importantly, the distribution of immunoreactive BDNF remained unchanged, as exemplified by its accumulation in mossy fiber terminals in the transgenic animals. GFP-labeled neurons could be readily visualized in distinct layers in the cerebral cortex where BDNF has been difficult to detect with currently available reagents. In the hippocampal formation, quantification of the GFP signal revealed that <10% of the neurons do not translate the Bdnf mRNA at detectable levels, with the highest proportion of strongly labeled neurons found in CA3.
| S-EPMC6957309 | biostudies-literature
Project description: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.
| S-EPMC3929946 | biostudies-literature
Project description:Fluorescent proteins (FPs) are indispensable markers for two-photon imaging of live tissue, especially in the brains of small model organisms. The quantity of physiologically relevant data collected, however, is limited by heat-induced damage of the tissue due to the high intensities of the excitation laser. We seek to minimize this damage by developing FPs with improved brightness. Among FPs with the same chromophore structure, the spectral properties can vary widely due to differences in the local protein environment. Using a physical model that describes the spectra of FPs containing the anionic green FP (GFP) chromophore, we predict that those that are blue-shifted in one-photon absorption will have stronger peak two-photon absorption cross sections. Following this prediction, we present 12 blue-shifted GFP homologues and demonstrate that they are up to 2.5 times brighter than the commonly used enhanced GFP (EGFP).
| S-EPMC5474692 | biostudies-literature
Project description:Semisynthetic green fluorescent proteins (GFPs) can be prepared by producing truncated GFPs recombinantly and assembling them with synthetic beta-strands of GFP. The yield from expressing the truncated GFPs is low, and the chromophore is either partially formed or not formed. An alternative method is presented in which full-length proteins are produced recombinantly with a protease site inserted between the structural element to be removed and the rest of the protein. The native peptide can then be replaced by cutting the protease site with trypsin, denaturing in guanidine hydrochloride to disrupt the complex, separating the native peptide from the rest of the protein by size exclusion, and refolding the protein in the presence of a synthetic peptide. We show that this method allows for removal and replacement of the interior chromophore containing helix and that the GFP barrel is capable of inducing chromophore formation in a synthetic interior helix.
| S-EPMC2783612 | biostudies-literature
Project description:Green fluorescent protein (GFP) and its derivatives have transformed the use and analysis of proteins for diverse applications. Like proteins, RNA has complex roles in cellular function and is increasingly used for various applications, but a comparable approach for fluorescently tagging RNA is lacking. Here, we describe the generation of RNA aptamers that bind fluorophores resembling the fluorophore in GFP. These RNA-fluorophore complexes create a palette that spans the visible spectrum. An RNA-fluorophore complex, termed Spinach, resembles enhanced GFP and emits a green fluorescence comparable in brightness with fluorescent proteins. Spinach is markedly resistant to photobleaching, and Spinach fusion RNAs can be imaged in living cells. These RNA mimics of GFP provide an approach for genetic encoding of fluorescent RNAs.
| S-EPMC3314379 | biostudies-literature
Project description:In the directed evolution experiments of EGFP or StayGold, the objective was to blue-shift the excitation light spectrum of GFP towards shorter wavelengths, resulting in an enhancement of fluorescence intensity upon 405 nm laser excitation. This facilitates the advancement of novel fluorescent proteins. These experiments represent a category of experiments that utilize REPLACE to achieve discontinuous directed evolution through FACS sorting (screening).
2024-09-30 | GSE271876 | GEO
Project description:The fluorescence of bacterial cells expressing a variant (GFPm) of the green fluorescent protein (GFP) was reduced to background levels by global replacement of the leucine residues of GFPm by 5,5,5-trifluoroleucine. Eleven rounds of random mutagenesis and screening via fluorescence-activated cell sorting yielded a GFP mutant containing 20 amino acid substitutions. The mutant protein in fluorinated form showed improved folding efficiency both in vivo and in vitro, and the median fluorescence of cells expressing the fluorinated protein was improved approximately 650-fold in comparison to that of cells expressing fluorinated GFPm. The success of this approach demonstrates the feasibility of engineering functional proteins containing many copies of abiological amino acid constituents.
| S-EPMC1955812 | biostudies-other