Biology subjects

Gonzalez, R. L.

Publications and source records attributed to Gonzalez, R. L..

28 records · Page 2Linked to original sources

bioRxiv2020

Bayesian inference: The comprehensive approach to analyzing single-molecule experiments

Biophysics experiments performed at single-molecule resolution contain exceptional insight into the structural details and dynamic behavior of biological systems. However, extracting this information from the corresponding experimental data unequivocally requires applying a biophysical model. Here, we discuss how to use probability theory to apply these models to single-molecule data. Many current single-molecule data analysis methods apply parts of probability theory, sometimes unknowingly, and thus miss out on the full set of benefits provided by this self-consistent framework. The full application of probability theory involves a process called Bayesian inference that fully accounts for the uncertainties inherent to single-molecule experiments. Additionally, using Bayesian inference provides a scientifically rigorous manner to incorporate information from multiple experiments into a single analysis and to find the best biophysical model for an experiment without the risk of overfitting the data. These benefits make the Bayesian approach ideal for analyzing any type of single-molecule experiment.

biophysics↗

bioRxiv2020

Structural insights into mRNA reading frame regulation by tRNA modification and slippery codon-anticodon pairing

Modifications in the tRNA anticodon, adjacent to the three-nucleotide anticodon, influence translation fidelity by stabilizing the tRNA to allow for accurate reading of the mRNA genetic code. One example is the N1-methylguaonosine modification at guanine nucleotide 37 (m1G37) located in the anticodon loop, immediately adjacent to the anticodon nucleotides 34-36. The absence of m1G37 in tRNAPro causes +1 frameshifting on polynucleotide, slippery codons. Here, we report structures of the bacterial ribosome containing tRNAPro bound to either cognate or slippery codons to determine how the m1G37 modification prevents mRNA frameshifting. The structures reveal that certain codon-anticodon contexts and m1G37 destabilize interactions of tRNAPro with the peptidyl site, causing large conformational changes typically only seen during EF-G mediated translocation of the mRNA-tRNA pairs. These studies provide molecular insights into how m1G37 stabilizes the interactions of tRNAPro with the ribosome and the influence of slippery codons on the mRNA reading frame. IMPACT STATEMENTChemical modifications near the tRNA anticodon and specific mRNA-tRNA pairs combine to control the ribosomal three-nucleotide mRNA reading frame, essential for the sequential addition of amino acids into polypeptide chains. Data depositionCrystallography, atomic coordinates, and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB codes 6NTA, 6NSH, 6NUO, 6NWY, 6O3M, 6OSI)

biochemistry↗

bioRxiv2020

Elongation Inhibitors do not Prevent the Release of Puromycylated Nascent Polypeptide Chains from Ribosomes

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.

biochemistry↗

bioRxiv2019

(p)ppGpp directly regulates translation initiation during entry into quiescence

Many bacteria exist in a state of metabolic quiescence where they must minimize energy consumption so as to maximize available resources over a potentially extended period of time. As protein synthesis is the most energy intensive metabolic process in a bacterial cell, it would be an appropriate target for downregulation during the transition from growth to quiescence. We find that when Bacillus subtilis exits growth, a subpopulation of cells emerges with very low levels of protein synthesis dependent on synthesis of the nucleotides (p)ppGpp. We show that (p)ppGpp inhibits protein synthesis in vivo and in vitro by preventing the allosteric activation of the essential GTPase Initiation Factor 2 (IF2) during translation initiation. Finally, we demonstrate that IF2 is an authentic in vivo target of (p)ppGpp during the entry into quiescence, thus providing a mechanistic basis for the observed attenuation of protein synthesis.

microbiology↗

bioRxiv2019

Multiplexed, bioorthogonal labeling of multicomponent, biomolecular complexes using genomically encoded, non-canonical amino acids

Stunning advances in the structural biology of multicomponent biomolecular complexes (MBCs) have ushered in an era of intense, structure-guided mechanistic and functional studies of these complexes. Nonetheless, existing methods to site-specifically conjugate MBCs with biochemical and biophysical labels are notoriously impracticable and/or significantly perturb MBC assembly and function. To overcome these limitations, we have developed a general, multiplexed method in which we genomically encode non-canonical amino acids (ncAAs) into multiple, structure-informed, individual sites within a target MBC; select for ncAA-containing MBC variants that assemble and function like the wildtype MBC; and site-specifically conjugate biochemical or biophysical labels to these ncAAs. As a proof-of-principle, we have used this method to generate unique single-molecule fluorescence resonance energy transfer (smFRET) signals reporting on ribosome structural dynamics that have thus far remained inaccessible to smFRET studies of translation.

bioengineering↗

bioRxiv2018

Recruitment of human eIF4G1 or DAP5 to the 5’ untranslated regions of a subset of cellular mRNAs drives cap-independent translation

During unfavorable human cellular conditions (e.g., tumor hypoxia, viral infection, etc.), canonical, cap-dependent mRNA translation is suppressed. Nonetheless, a subset of physiologically important mRNAs (e.g., HIF-1, FGF-9, and p53) is still translated by an unknown, cap-independent mechanism. Additionally, expression levels of eIF4G and its homolog, death associated protein 5 (DAP5), are elevated. Using fluorescence anisotropy binding studies, luciferase reporter-based in vitro translation assays, and mutational analyses, here we demonstrate that eIF4GI and DAP5 specifically bind to the 5 UTRs of these cap-independently translated mRNAs. Surprisingly, we find that the eIF4E binding domain of eIF4GI increases not only the binding affinity, but also the selectivity among these mRNAs. We further demonstrate that the affinities of eIF4GI and DAP5 binding to these 5 UTRs correlate with the efficiency with which these factors drive cap-independent translation of these mRNAs. Integrating the results of our binding and translation assays, we show that eIF4GI and/or DAP5 are critical for recruitment of a specific subset of mRNAs to the ribosome and provide mechanistic insight into their cap-independent translation.

biophysics↗

bioRxiv2018

Bayesian-estimated hierarchical HMMs enable robust analysis of single-molecule kinetic heterogeneity

Single-molecule kinetic experiments allow the reaction trajectories of individual biomolecules to be directly observed, eliminating the effects of population averaging and providing a powerful approach for elucidating the kinetic mechanisms of biomolecular processes. A major challenge to the analysis and interpretation of these experiments, however, is the kinetic heterogeneity that almost universally complicates the recorded single-molecule signal versus time trajectories (i.e., signal trajectories). Such heterogeneity manifests as changes and/or differences in the transition rates that are observed within individual signal trajectories or across a population of signal trajectories. Although characterizing kinetic heterogeneity can provide critical mechanistic information, there are currently no computational methods available that effectively and/or comprehensively enable such analysis. To address this gap, we have developed a computational algorithm and software program, hFRET, that uses the variational approximation for Bayesian inference to estimate the parameters of a hierarchical hidden Markov model, thereby enabling robust identification and characterization of kinetic heterogeneity. Using simulated signal trajectories, we demonstrate the ability of hFRET to accurately and precisely characterize kinetic heterogeneity. In addition, we use hFRET to analyze experimentally recorded signal trajectories reporting on the conformational dynamics of ribosomal pre-translocation (PRE) complexes. The results of our analyses demonstrate that PRE complexes exhibit kinetic heterogeneity, reveal the physical origins of this heterogeneity, and allow us to expand the current model of PRE complex dynamics. The methods described here can be applied to signal trajectories generated using any type of signal and can be easily extended to the analysis of signal trajectories exhibiting more complex kinetic behaviors. Moreover, variations of our approach can be easily developed to integrate kinetic data obtained from different experimental constructs and/or from molecular dynamics simulations of a biomolecule of interest. The hFRET source code, graphical user interface, and user manual can be downloaded as freeware at https://github.com/GonzalezBiophysicsLab/hFRET.

biophysics↗

bioRxiv2018

Real-time structural dynamics of late steps in bacterial translation initiation visualized using time-resolved cryogenic electron microscopy

Bacterial translation initiation entails the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex (IC) to form a 70S IC that subsequently matures into a 70S elongation-competent complex (70S EC). Rapid and accurate 70S IC formation is promoted by 30S IC-bound initiation factor (IF) 1 and the guanosine triphosphatase (GTPase) IF2, both of which must ultimately dissociate from the 70S IC before the resulting 70S EC can begin translation elongation1. Although comparison of 30S2-6 and 70S5,7-9 IC structures have revealed that the ribosome, IFs, and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during 70S IC formation, structures of any intermediates formed during these rearrangements, and contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, lack of an authentic 70S EC structure has precluded an understanding of ribosome, IF, and fMet-tRNAfMet rearrangements that occur upon maturation of a 70S IC into a 70S EC. Using time-resolved cryogenic electron microscopy (TR cryo-EM)10 we report the first, near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, IF dissociation, and fMet-tRNAfMet positioning during 70S EC formation. We have found that, within ~20-80 ms, rearrangements of the 30S subunit and IF2, uniquely captured in its GDP*Pi-bound state, stabilize fMet-tRNAfMet in its intermediate, 70S P/I, configuration7 and trigger dissociation of IF1 from the 70S IC. Within the next several hundreds of ms, dissociation of IF2 from the 70S IC is coupled to further remodeling of the ribosome that positions fMet-tRNAfMet into its final, P/P, configuration within the 70S EC. Our results demonstrate the power of TR cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.

molecular biology↗

bioRxiv2018

D-amino acid-mediated translation arrest is modulated by the identity of the incoming aminoacyl-tRNA

A complete understanding of the determinants that restrict D-amino acid incorporation by the ribosome, which is of interest to both basic biologists as well as the protein engineering community, remains elusive. Previously, we demonstrated that D-amino acids are successfully incorporated into the C-terminus of the nascent polypeptide chain. Ribosomes carrying the resulting peptidyl-D-aminoacyl-tRNA (peptidyl-D-aa-tRNA) donor substrate, however, partition into subpopulations that either undergo translation arrest through inactivation of the ribosomal peptidyl-transferase center (PTC) or remain translationally competent. The proportion of each subpopulation is determined by the identity of the D-amino acid sidechain. Here, we demonstrate that the identity of the aminoacyl-tRNA (aa-tRNA) acceptor substrate that is delivered to ribosomes carrying a peptidyl-D-aa-tRNA donor further modulates this partitioning. Our discovery demonstrates that it is the pairing of the peptidyl-D-aa-tRNA donor and the aa-tRNA acceptor that determines the activity of the PTC. Moreover, we provide evidence that both the amino acid and tRNA components of the aa-tRNA donor contribute synergistically to the extent of arrest. The results of this work deepen our understanding of the mechanism of D-amino acid-mediated translation arrest and how cells avoid this precarious obstacle, reveal similarities to other translation arrest mechanisms involving the PTC, and provide a new route for improving the yields of engineered proteins containing D-amino acids.

biochemistry↗

bioRxiv2017

Temporal super-resolution in single-molecule experiments

Many time-resolved, single-molecule biophysics experiments seek to characterize the kinetics of biomolecular systems exhibiting dynamics that challenge the time resolution of the given technique. Here we present a general, computational approach to this problem that employs Bayesian inference to learn the underlying dynamics of such systems, even when they are much faster than the time resolution of the experimental technique being used. By accurately and precisely inferring rate constants, our Bayesian Inference for the Analysis of Sub-temporal-resolution Data (BIASD) approach effectively enables the experimenter to super-resolve the poorly resolved dynamics that are present in their data.

biophysics↗