Ongoing improvements of genetically encoded fluorescent proteins have enhanced cellular localization studies and performance of biosensors, such as environmentally or mechanically sensitive fluorescence resonance energy transfer pairs, in cell biological and biophysical research. The brightest yellow fluorescent protein, widely used in these studies is YPet, derived from the jellyfish Aequorea victoria via the GFP derivative Venus. YPet dimerizes at concentrations used in cellular studies (KD1-2 = 3.4 μM) which impacts quantitative interpretation of emission intensity, rotational freedom, energy transfer, and lifetime. Although YPet is nearly 30% brighter than Venus, no atomic structures of YPet have been reported to ascertain the structural differences leading to the higher brightness, possibly due to the tendency to dimerize or oligomerize. Here, we report properties of a new YPet derivative, mCLIFY, a monomeric, bright, yellow, and long-lived fluorescent protein created by circular permutation of YPet and substitution of the amino acid residues thought to mediate dimerization. mCLIFY retains the advantageous photophysical properties of YPet but does not dimerize at least up to 40 μM concentration. We determined the atomic structure of mCLIFY at 1.57-Å resolution. Extensive characterization of the photophysical and structural properties of YPet and mCLIFY allowed us to elucidate the bases of their long lifetimes, enhanced brightness, and the difference in propensity to dimerize.

Premature termination codon (PTC) diseases account for ∼12% of all human disease mutations. Although there are no FDA approved treatments for increasing PTC readthrough, one readthrough inducing drug, ataluren, has conditional approval for treatment of Duchenne muscular dystrophy elsewhere. Ataluren displays low toxicity in clinical trials for treatment of PTC diseases, but its therapeutic effects are inconsistent. The messenger RNA (mRNA) sequence context of a PTC is a major determinant of PTC readthrough efficiency. We have shown that ataluren stimulates readthrough exclusively by competitively inhibiting release factor complex (RFC) catalysis of translation termination. Here, using an in vitro reconstituted system, we demonstrate that PTC identity and the immediately adjacent mRNA sequence contexts modulate RFC activity in terminating peptide elongation. Such modulation largely determines the effectiveness of ataluren in stimulating readthrough, whether added alone or in combination with either the aminoglycoside G418 or an anticodon edited aa-tRNA, which stimulate readthrough by mechanisms orthogonal to that of ataluren. Our results suggest a potential rationale for the variability of ataluren effectiveness in stimulating readthrough. We hypothesize that patients harboring a PTC mutation within a sequence context promoting strong interaction with RFC will be resistant to ataluren, but that ataluren treatment will be more effective for patient sequences conferring weaker interaction with RFC.

Many genetic disorders are caused by premature stop codon (PSC) mutations, but only one TRID (Translational readthrough-inducing drug), ataluren, has been approved for clinical use. Recently, we used single-molecule TIRF with a cell-free in vitro assay and found that ataluren competitively inhibits productive release factor complex (RFC, eRF1.eRF3.GTP) binding to the pre-termination complex. We found that such inhibition occurs before or at the peptidyl-tRNA hydrolysis step (Huang et al., Nat. Comm., 2022, 13: 2413). Here we report new results using an sm-FRET assay with a Cy3-labeled peptidyl-tRNA bound in the ribosomal P-site adjacent to a UGA stop codon in the A-site and Cy5-labeled human eRF1. Upon RFC binding, we observe transient FRET efficiency, E = ∼0.25, between Cy5-eRF1 and Cy3-tRNA, consistent with the successful accommodation of eRF1 within the A-site. Following peptidyl-tRNA hydrolysis, we find a strong correlation between eRF1 and tRNA dissociation times, unlike the weak correlation between peptide release and tRNA dissociation times that we observed previously (Huang et al., ibid.). Additionally, we found that ataluren and added near-cognate suppressor tRNA each had a significant inhibitory effect on the arrival time of RFCs at the ribosome, and that ataluren in combination with the aminoglycoside G418 had an even stronger inhibitory effect compared with ataluren or G418 alone. These results support our earlier suggestion (Ng et al., PNAS, 2021, 118: e2020599118) that ataluren or ataluren-like TRIDs could potentiate the therapeutic effects of aminoglycosides on PSC diseases. We expect that our ongoing studies will aid in elucidating the readthrough mechanisms of other TRIDs leading to enhanced and safer treatments of PSC diseases.

Fluorescent proteins (FP) have become widely used biophysical and cell biological tools that report on a variety of processes within cells. The popular yellow FP, YPet, serves as either a donor or acceptor for many FRET-based biosensors, yet it has been difficult to fully characterize. In studying our circularly permuted and equally bright variant, mCLIFY, features common to YPet were revealed, including β-barrel and chromophore sequence homology. We determined the atomic structure of mCLIFY to 1.6 Å resolution, which reveals a remarkably well-aligned π-interaction between the chromophore with Tyr37, as well as a water-mediated network of hydrogen bonds which supports the chromophore. Both features are likely promoting the robust photophysical properties of mCLIFY. To better understand the self-association properties of YPet, known to be a weak dimer, sedimentation velocity analytical ultracentrifugation (SV-AUC) determined a monomer-dimer dissociation constant KD of 3.4 μM, whereas mCLIFY did not dimerize. Size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering and multi-angle light scattering (SEC-SAXS-MALS) confirmed these findings. SAXS studies show that mCLIFY correlates well to the solution monomer, whereas YPET exists as an antiparallel dimer in solution. Their fluorescence intensities and anisotropies were compared via FLIM (Fluorescence Lifetime Imaging Microscopy) of FPs expressed in E. coli. Protein concentrations increased above 5 μM for both FPs within 45 minutes of IPTG induction. Unexpectedly, average anisotropy of YPet significantly decreased from 0.322 to 0.183 with increasing protein concentration presumably due to HOMO-FRET and/or trivial reabsorption of emitted photons. Anisotropy for mCLIFY (0.316) decreased at much higher concentrations. The data presented here suggest that potential quantitative errors from overexpression of non-monomeric FPs may be avoided with newly available, more fully studied monomeric variants like mCLIFY.

Significance Elongation factor G (EF-G) uses energy stored in GTP to catalyze movement of transfer RNAs and messenger RNA in the ribosome during the translocation step of prokaryotic protein synthesis. Using single-molecule polarized fluorescence microscopy, three-dimensional rotational motions of individual domains of EF-G were directly captured, for the first time to our knowledge, during normal translocation. Our observations strongly imply a hybrid model, in which the initial steps of translocation are ribosome unlocking driven by a force generated via EF-G–dependent GTP hydrolysis, and further steps of translocation are mainly driven by the energetics of the ribosome itself. These results demonstrate that the ribosome and EF-G make use of power-stroke and Brownian-ratchet mechanisms to ensure efficiency and accuracy of translocation.

During the elongation cycle of protein synthesis, translocation of tRNAs and mRNA is catalyzed by the GTPase elongation factor G (EF-G) with high precision and speed. Conversion of the GTP to the GDP form of EF-G is considered essential for translocation, but the structural dynamics on the ribosome have not been reported. We used single molecule polarized total internal reflection fluorescence (polTIRF) microscopy to characterize tilting and rotational fluctuations within specific domains of EF-G. When EF-G binds to the ribosomal pre-translocation (PRE) complex, domains I and IV of EF-G undergo small rotations (10-15°) in conjunction with translocation, whereas domain III shows a much greater angular change, averaging 50°. Viomycin (Vio), which prevents translocation, reduces the rotational motions of domain III to 10-15° but has virtually no effect on the other domains. Spectinomycin also reduces domain III motions but less strongly than Vio. EF-G binding to ribosomal initiation complexes lacking A-site tRNA gives a similar pattern of domain rotations, but with shorter dwell times. In this case, the large rotation of domain III is barely inhibited by Vio. Irrespective of completion of translocation or presence of A-site tRNA, the initial 10-15° rotations of EF-G domains I, III and IV in the ribosome/EF-G complex indicate that the EF-G initially shifts the minimum of the free energy profile in the direction of translocation, suggesting that EF-G generates a force on the ribosome and/or the mRNA and tRNAs. Near the end of translocation, domain III completes its rotation either to push the mRNA and tRNAs (a working stroke) or to prevent reversal of translocation driven by thermal fluctuations (a ratchet). Supported by NIH grant GM080376 to YEG and BSC and AHA fellowship 12POST8910014 to CC.

Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification.

of BipA using amide hydrogen/deuterium exchange mass spectrometry (HDXMS). These data indicate that GTPand ppGpp-binding lead to large scale conformational changes that are propagated throughout BipA, underscoring the idea that BipA is a metastable molecule where mutually exclusive association of GTP or ppGpp drive equilibria to alternate distinct conformations resulting differential ribosome binding. Molecular dynamics simulations together with covariance analysis are being used to explore the dynamic allostery between the GTPase and novel C-terminal domain.

translation are not well understood. Previously, we demonstrated that solventaccessible volume surrounding a modifiable cysteine increases monotonically with increase in the van der Waal’s volume of the adjacent side chain (Lu et al., J.Mol.Biol. 411: 499-510, 2011) and that the magnitude of this effect depends on location within the ribosomal tunnel. using a photocrosslinking approach, we confirm these results. We extend these studies to investigate whether mutations in the nascent peptide deep in the tunnel affect the accessibility of a modifiable reporter cysteine at the exit port and whether specific regions of the tunnel instigate these effects. Tryptophan vis-a-vis alanine, engineered into the nascent peptide at a distance of 17-19 residues from the PTC, alters the accessibility of residues at the exit port, a distance of 33 residues from the PTC, roughly 50 angstroms from the introduced point mutations. These findings are consistent with long-range rearrangements and may contribute to mechanisms governing sequence-specific signaling from different regions of the tunnel during translation. Supported by NIH grant R01GM52302.

Ribosomal synthesis of proteins proceeds with pauses that regulate the rhythm of protein synthesis. In order to study the factors that control translation rates, we use the expression of fast maturing Emerald Green Fluorescent Protein (EmGFP) by a reconsitituted E. coli cell-free translation system. In order to quantify translation rate in identfied short segments of the sequence, the existing ribosomes and Phe-tRNAPhe in the cell-free mixture are replaced by fluorescent labeled Phe-tRNAPhe(Cy5.5) and L11(Cy3)-ribosomes. Single-molecule FRET trajectories report of multiple accommodations of Phe-tRNAPhes on single ribosomes during synthesis of EmGFP. An algorithm was developed to identify FRET pulses objectively by anti-correlation of donor and acceptor intensities. The time intervals between two consecutive Phe-tRNAPhe(Cy5.5) FRET pulses can be assigned to particular sequence segments according to their timing relative to two characteristic Phe-Phe doublets near the middle of the EmGFP sequence. Translation proceeds with variable rates which are correlated to codon and isoacceptor tRNA usage. Codon CGG, coding for a rare tRNAArg, slows elongation approximately 5-fold compared with CGC, coding for a more plentiful tRNAArg. This difference is eliminated when the total concentration of tRNAArg isoacceptors is increased. These results quantify the regulation of elongation by tRNA availability. Decreased translational rate due to tRNA selection is concomitant with the emergence of an upstream nascent polypeptide from the ribosomal exit tunnel. Thus, the rhythm of translation can have an upstream impact on co-translational processes such as protein folding. Supported by NIH Grant GM080376 and HFSP.

We present a flexible, real-time-coupled transcription-translation assay that involves the continuous monitoring of fluorescent Emerald GFP formation. Along with numerical simulation of a reaction kinetics model, the assay permits quantitative estimation of the effects on full-length protein synthesis of various additions, subtractions or substitutions to the protein synthesis machinery. Since the assay uses continuous fluorescence monitoring, it is much simpler and more rapid than other assays of protein synthesis and is compatible with high-throughput formats. Straightforward alterations of the assay permit determination of (i) the fraction of ribosomes in a cell-free protein synthesis kit that is active in full-length protein synthesis and (ii) the relative activities in supporting protein synthesis of modified (e.g. mutated, fluorescent-labeled) exogenous components (ribosomes, amino acid-specific tRNAs) that replace the corresponding endogenous components. Ribosomes containing fluorescent-labeled L11 and tRNAs labeled with fluorophores in the D-loop retain substantial activity. In the latter case, the extent of activity loss correlates with a combination of steric bulk and hydrophobicity of the fluorophore.