Inference of evolutionary jumps in large phylogenies using Lévy processes

Although it is now widely accepted that the rate of phenotypic evolution may not necessarily be constant across large phylogenies, the frequency and phylogenetic position of periods of rapid evolution remain unclear. In his highly influential view of evolution, G. G. Simpson supposed that such evolutionary jumps occur when organisms transition into so-called new adaptive zones, for instance after dispersal into a new geographic area, after rapid climatic changes, or following the appearance of an evolutionary novelty. Only recently, large, accurate and well calibrated phylogenies have become available that allow testing this hypothesis directly, yet inferring evolutionary jumps remains computationally very challenging. Here, we develop a computationally highly efficient algorithm to accurately infer the rate and strength of evolutionary jumps as well as their phylogenetic location. Following previous work we model evolutionary jumps as a compound process, but introduce a novel approach to sample jump configurations that does not require matrix inversions and thus naturally scales to large trees. We then make use of this development to infer evolutionary jumps in Anolis lizards and Loriinii parrots where we find strong signal for such jumps at the basis of clades that transitioned into new adaptive zones, just as postulated by Simpson’s hypothesis

Recognition of G-1:C73 Atomic Groups by <i>Escherichia </i><i>c</i><i>oli</i> Histidyl-tRNA Synthetase

This work focuses on the RNA−protein interactions necessary for efficient aminoacylation of tRNAHis by Escherichia coli histidyl-tRNA synthetase (HisRS). The E. coli tRNAHis acceptor stem is characterized by a unique “extra” G-1:C73 base pair. Previous in vivo and in vitro studies showed that G-1:C73 is a major recognition element for E. coli HisRS. To further probe the role of the G-1:C73 base pair in specific aminoacylation, we carried out atomic group “mutagenesis” studies. Systematic base analogue substitutions at the −1:73 position of chemically synthesized microhelixHis substrates suggest that the G-1 base serves to position the 5‘-monophosphate, which is critical for aminoacylation. Additionally, the C73 and G-1 bases contain major groove exocyclic atomic groups that contribute to HisRS recognition

Substrate and Enzyme Functional Groups Contribute to Translational Quality Control by Bacterial Prolyl-tRNA Synthetase

Aminoacyl-tRNA synthetases activate specific amino acid substrates and attach them via an ester linkage to cognate tRNA molecules. In addition to cognate proline, prolyl-tRNA synthetase (ProRS) can activate cysteine and alanine and misacylate tRNA^Pro. Editing of the misacylated aminoacyl-tRNA is required for error-free protein synthesis. An editing domain (INS) appended to bacterial ProRS selectively hydrolyzes Ala-tRNA^Pro, whereas Cys-tRNA^Pro is cleared by a freestanding editing domain, YbaK, through a unique mechanism involving substrate sulfhydryl chemistry. The detailed mechanism of catalysis by INS is currently unknown. To understand the alanine specificity and mechanism of catalysis by INS, we have explored several possible mechanisms of Ala-tRNA^Pro deacylation via hybrid QM/MM calculations. Experimental studies were also performed to test the role of several residues in the INS active site as well as various substrate functional groups in catalysis. Our results support a critical role for the tRNA 2′-OH group in substrate binding and catalytic water activation. A role is also proposed for the protein’s conserved GXXXP loop in transition state stabilization and for the main chain atoms of Gly261 in a proton relay that contributes substantially to catalysis

Single-Molecule Spectroscopic Study of Dynamic Nanoscale DNA Bending Behavior of HIV-1 Nucleocapsid Protein

We have studied the conformational dynamics associated with the nanoscale DNA bending induced by human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein using single-molecule Förster resonance energy transfer (SM-FRET). To gain molecular-level insights into how the HIV-1 NC locally distorts the structures of duplexed DNA segments, the dynamics, reversibility, and sequence specificity of the DNA bending behavior of NC have been systematically studied. We have performed SM-FRET measurements on a series of duplexed DNA segments with varying sequences, lengths, and local structures in the presence of the wide-type HIV-1 NC and NC mutants lacking either the basic N-terminal domain or the zinc fingers. On the basis of the SM-FRET results, we have proposed a possible mechanism for the NC-induced DNA bending in which both NC’s zinc fingers and N-terminal domain are found to play crucial roles. The SM-FRET results reported here add new mechanistic insights into the biological behaviors and functions of HIV-1 NC as a retroviral DNA-architectural protein which may play critical roles in the compaction, nuclear import, and integration of the proviral DNA during the retroviral life cycle

Wild-Type RNA Microhelix^Ala and 3:70 Variants: Molecular Dynamics Analysis of Local Helical Structure and Tightly Bound Water

Molecular dynamics simulations of RNA microhelixAla indicate that G:U and other 3:70 purine:pyrimidine wobble pairs induce local deviations from A-form geometry in their respective microhelices; the helix is underwound at the base-pair step above and overwound at the base-pair step below, in each case by about 7−9° compared to canonical A-form RNA. On the basis of analysis of average water densities and residence lifetimes, the wild-type microhelix strongly binds a water molecule in the minor groove of the 3:70 base pair, consistent with crystallographic analyses of an RNA duplex derived from the acceptor stem of Escherichia coli tRNAAla. Other wobble pairs show water binding at this position but to a lesser degree; the strength of water binding correlates directly with the measured aminoacylation activities of the microhelices as substrates for E. coli alanyl-tRNA synthetase (G:U > 2AA:IsoC > G:dU > I:U). Watson−Crick base pairs at the 3:70 position show no tendency toward specific hydration. This tightly bound minor-groove water in the microhelices with 3:70 wobble pairs evidently does not function to stabilize a particular local helical structure, but it may play a role as a specific recognition element or serve as an indicator of interaction specificity between the microhelix and a hydrogen-bonding residue of the aminoacyl-tRNA synthetase

Aminoacyl-tRNA Substrate and Enzyme Backbone Atoms Contribute to Translational Quality Control by YbaK

Amino acids are covalently attached to their corresponding transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases. Proofreading mechanisms exist to ensure that high fidelity is maintained in this key step in protein synthesis. Prolyl-tRNA synthetase (ProRS) can misacylate cognate tRNA^Pro with Ala and Cys. The <i>cis</i>-editing domain of ProRS (INS) hydrolyzes Ala-tRNA^Pro, whereas Cys-tRNA^Pro is hydrolyzed by a single domain editing protein, YbaK, <i>in trans</i>. Previous studies have proposed a model of substrate-binding by bacterial YbaK and elucidated a substrate-assisted mechanism of catalysis. However, the microscopic steps in this mechanism have not been investigated. In this work, we carried out biochemical experiments together with a detailed hybrid quantum mechanics/molecular mechanics study to investigate the mechanism of catalysis by Escherichia coli YbaK. The results support a mechanism wherein cyclization of the substrate Cys results in cleavage of the Cys-tRNA ester bond. Protein side chains do not play a significant role in YbaK catalysis. Instead, protein backbone atoms play crucial roles in stabilizing the transition state, while the product is stabilized by the 2′-OH of the tRNA

Functional Role of the Prokaryotic Proline-tRNA Synthetase Insertion Domain in Amino Acid Editing^†

Aminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to cognate tRNAs in a two-step process that is critical for the faithful translation of genetic information. During the first chemical step of tRNA aminoacylation, noncognate amino acids that are smaller than or isosteric with the cognate substrate can be misactivated. Thus, to maintain high accuracy during protein translation, some synthetases have evolved an editing mechanism. Previously, we showed that class II Escherichia coli proline-tRNA synthetase (ProRS) is capable of (1) weakly misactivating Ala, (2) hydrolyzing the misactivated Ala-AMP in a reaction known as pretransfer editing, and (3) deacylating a mischarged Ala-tRNAPro variant via a post-transfer editing pathway. In contrast to most systems where an editing function has been established, pretransfer editing by E. coli ProRS occurs in a tRNA-independent fashion. However, neither the pre- nor the post-transfer editing active site(s) has been identified. Sequence analyses revealed that most prokaryotic ProRSs possess a large insertion domain (INS) between class II conserved motifs 2 and 3. The function of the ∼180-amino acid INS in E. coli ProRS is the subject of this investigation. Alignment-guided Ala scanning mutagenesis was carried out to test conserved amino acid residues present in the INS for their role in pre- and post-transfer editing. Our biochemical data and modeling studies suggest that the prokaryotic INS plays a critical role in editing and that this activity resides in a domain that is functionally and structurally distinct from the aminoacylation active site

Single-Molecule Study of the Inhibition of HIV-1 Transactivation Response Region DNA/DNA Annealing by Argininamide

Single-molecule spectroscopy was used to examine how a model inhibitor of HIV-1, argininamide, modulates the nucleic acid chaperone activity of the nucleocapsid protein (NC) in the minus-strand transfer step of HIV-1 reverse transcription, in vitro. In minus-strand transfer, the transactivation response region (TAR) RNA of the genome is annealed to the complementary “TAR DNA” generated during minus-strand strong-stop DNA synthesis. Argininamide and its analogs are known to bind to the hairpin bulge region of TAR RNA as well as to various DNA loop structures, but its ability to inhibit the strand transfer process has only been implied. Here, we explore how argininamide modulates the annealing kinetics and secondary structure of TAR DNA. The studies reveal that the argininamide inhibitory mechanism involves a shift of the secondary structure of TAR, away from the NC-induced “Y” form, an intermediate in reverse transcription, and toward the free closed or “C” form. In addition, more potent inhibition of the loop-mediated annealing pathway than stem-mediated annealing is observed. Taken together, these data suggest a molecular mechanism wherein argininamide inhibits NC-facilitated TAR RNA/DNA annealing in vitro by interfering with the formation of key annealing intermediates

Structure of Tetrahelical DNA Homopolymers Supports Quadruplex World Hypothesis

We previously reported a tetrahelical monomolecular architecture of DNA, tmDNA, which employs G-quartets and an all-parallel GGGTGGGTGGGTGGG (G3T) quadruplex as the repeating unit. Based on thermodynamic and kinetic studies, we proposed that covalently joined (G3T)n units formed an uninterrupted programmable homopolymer; however, structural evidence for the tmDNA architecture was lacking. Here, we used NMR spectroscopy of wild-type and single-inosine-substituted constructs to characterize both monomolecular (G3T)2 and bimolecular quadruplex-Mg-coupled versions of tmDNA. The NMR results support an architecture consisting of uninterrupted stacked G-tetrads in both the monomolecular constructs and bimolecular assemblies. Taken together, these data support the formation of a stable programmable homopolymeric tmDNA architecture, which may have been a precursor to the modern-day Watson–Crick DNA duplex

Single-Molecule Fluorescence Using Nucleotide Analogs: A Proof-of-Principle

Fluorescent nucleotide analogues, such as 2-aminopurine (2AP) and pyrrolo-C (PyC), have been extensively used to study nucleic acid local conformational dynamics in bulk experiments. Here we present a proof-of-principle approach using 2AP and PyC fluorescence at the single-molecule level. Our data show that ssDNA, dsDNA, or RNA containing both 2AP and PyC can be monitored using single-molecule fluorescence and a click chemistry immobilization method. We demonstrate that this approach can be used to monitor DNA and RNA in real time. This is the first reported assay using fluorescent nucleotide analogs at the single-molecule level. We anticipate that single 2AP or PyC fluorescence will have numerous applications in studies of DNA and RNA, including protein-induced base-flipping dynamics in protein–nucleic acid complexes