The 3-Minihelix tRNA Evolution Theorem.

Transfer RNA (tRNA) is the central intellectual property in the evolution of life on Earth. tRNA evolved from repeats and inverted repeats of known sequence. The anticodon and the T stem-loop-stems are homologs with significant conserved sequence identity. A number of models have been advanced to explain tRNA evolution. No 2-minihelix model or accretion model (built a stem at a time) can be correct, in part because of anticodon and T stem-loop-stem identity. Only a 3-minihelix model is adequate.

Homology threading to generate RNA polymerase structures.

Homology threading is a powerful technology for generating structural models based on homologous structures. Here we use threading to generate four complex RNA polymerase models. The models appear to be as useful as x-ray crystal structures or cryo-electron microscopy structures to support research projects.

Ribosome Structure, Function, and Early Evolution.

Ribosomes are among the largest and most dynamic molecular motors. The structure and dynamics of translation initiation and elongation are reviewed. Three ribosome motions have been identified for initiation and translocation. A swivel motion between the head/beak and the body of the 30S subunit was observed. A tilting dynamic of the head/beak versus the body of the 30S subunit was detected using simulations. A reversible ratcheting motion was seen between the 30S and the 50S subunits that slide relative to one another. The 30S⁻50S intersubunit contacts regulate translocation. IF2, EF-Tu, and EF-G are homologous G-protein GTPases that cycle on and off the same site on the ribosome. The ribosome, aminoacyl-tRNA synthetase (aaRS) enzymes, transfer ribonucleic acid (tRNA), and messenger ribonucleic acid (mRNA) form the core of information processing in cells and are coevolved. Surprisingly, class I and class II aaRS enzymes, with distinct and incompatible folds, are homologs. Divergence of class I and class II aaRS enzymes and coevolution of the genetic code are described by analysis of ancient archaeal species.

Type-II tRNAs and Evolution of Translation Systems and the Genetic Code.

Because tRNA is the core biological intellectual property that was necessary to evolve translation systems, tRNAomes, ribosomes, aminoacyl-tRNA synthetases, and the genetic code, the evolution of tRNA is the core story in evolution of life on earth. We have previously described the evolution of type-I tRNAs. Here, we use the same model to describe the evolution of type-II tRNAs, with expanded V loops. The models are strongly supported by inspection of typical tRNA diagrams, measuring lengths of V loop expansions, and analyzing the homology of V loop sequences to tRNA acceptor stems. Models for tRNA evolution provide a pathway for the inanimate-to-animate transition and for the evolution of translation systems, the genetic code, and cellular life.

Five checkpoints maintaining the fidelity of transcription by RNA polymerases in structural and energetic details.

Transcriptional fidelity, which prevents the misincorporation of incorrect nucleoside monophosphates in RNA, is essential for life. Results from molecular dynamics (MD) simulations of eukaryotic RNA polymerase (RNAP) II and bacterial RNAP with experimental data suggest that fidelity may involve as many as five checkpoints. Using MD simulations, the effects of different active site NTPs in both open and closed trigger loop (TL) structures of RNAPs are compared. Unfavorable initial binding of mismatched substrates in the active site with an open TL is proposed to be the first fidelity checkpoint. The leaving of an incorrect substrate is much easier than a correct one energetically from the umbrella sampling simulations. Then, the closing motion of the TL, required for catalysis, is hindered by the presence of mismatched NTPs. Mismatched NTPs also lead to conformational changes in the active site, which perturb the coordination of magnesium ions and likely affect the ability to proceed with catalysis. This step appears to be the most important checkpoint for deoxy-NTP discrimination. Finally, structural perturbations in the template DNA and the nascent RNA in the presence of mismatches likely hinder nucleotide addition and provide the structural foundation for backtracking followed by removing erroneously incorporated nucleotides during proofreading.

Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation.

To study fidelity of RNA polymerase II (Pol II), we analyzed properties of the 6-azauracil-sensitive and TFIIS-dependent E1103G mutant of rbp1 (rpo21), the gene encoding the catalytic subunit of Pol II in Saccharomyces cerevisiae. Using an in vivo retrotransposition-based transcription fidelity assay, we observed that rpb1-E1103G causes a 3-fold increase in transcription errors. This mutant showed a 10-fold decrease in fidelity of transcription elongation in vitro. The mutation does not appear to significantly affect translocation state equilibrium of Pol II in a stalled elongation complex. Primarily, it promotes NTP sequestration in the polymerase active center. Furthermore, pre-steady-state analyses revealed that the E1103G mutation shifted the equilibrium between the closed and the open active center conformations toward the closed form. Thus, open conformation of the active center emerges as an intermediate essential for preincorporation fidelity control. Similar mechanisms may control fidelity of DNA-dependent DNA polymerases and RNA-dependent RNA polymerases.

The RNA polymerase bridge helix YFI motif in catalysis, fidelity and translocation.

The bridge α-helix in the ß' subunit of RNA polymerase (RNAP) borders the active site and may have roles in catalysis and translocation. In Escherichia coli RNAP, a bulky hydrophobic segment near the N-terminal end of the bridge helix is identified (ß' 772-YFI-774; the YFI motif). YFI is located at a distance from the active center and adjacent to a glycine hinge (ß' 778-GARKG-782) involved in dynamic bending of the bridge helix. Remarkably, amino acid substitutions in YFI significantly alter intrinsic termination, pausing, fidelity and translocation of RNAP. F773V RNAP largely ignores the λ tR2 terminator at 200µM NTPs and is strongly reduced in λ tR2 recognition at 1µM NTPs. F773V alters RNAP pausing and backtracking and favors misincorporation. By contrast, the adjacent Y772A substitution increases fidelity and exhibits other transcriptional defects generally opposite to those of F773V. All atom molecular dynamics simulation revealed two separate functional connections emanating from YFI explaining the distinct effects of substitutions: Y772 communicates with the active site through the link domain in the ß subunit, whereas F773 communicates through the fork domain in the ß subunit. I774 interacts with the F-loop, which also contacts the glycine hinge of the bridge helix. These results identified negative and positive circuits coupled at YFI and employed for regulation of catalysis, elongation, termination and translocation.

Energetic and structural details of the trigger-loop closing transition in RNA polymerase II.

An evolutionarily conserved element in RNA polymerase II, the trigger loop (TL), has been suggested to play an important role in the elongation rate, fidelity of selection of the matched nucleoside triphosphate (NTP), catalysis of transcription elongation, and translocation in both eukaryotes and prokaryotes. In response to NTP binding, the TL undergoes large conformational changes to switch between distinct open and closed states to tighten the active site and avail catalysis. A computational strategy for characterizing the conformational transition pathway is presented to bridge the open and closed states of the TL. Information from a large number of independent all-atom molecular dynamics trajectories from Hamiltonian replica exchange and targeted molecular dynamics simulations is gathered together to assemble a connectivity map of the conformational transition. The results show that with a cognate NTP, TL closing should be a spontaneous process. One major intermediate state is identified along the conformational transition pathway, and the key structural features are characterized. The complete pathway from the open TL to the closed TL provides a clear picture of the TL closing.

Peak Cenozoic warmth enabled deep-sea sand deposition.

The early Eocene (~ 56-48 million years ago) was marked by peak Cenozoic warmth and sea levels, high CO2, and largely ice-free conditions. This time has been described as a period of increased continental erosion and silicate weathering. However, these conclusions are based largely on geochemical investigation of marine mudstones and carbonates or study of intermontane Laramide basin settings. Here, we evaluate the marine coarse siliciclastic response to early Paleogene hothouse climatic and oceanographic conditions. We compile an inventory of documented sand-rich (turbidite) deep-marine depositional systems, recording 59 instances of early Eocene turbidite systems along nearly all continental margins despite globally-elevated sea levels. Sand-rich systems were widespread on active margins (42 instances), but also on passive margins (17 instances). Along passive margins, 13 of 17 early Eocene systems are associated with known Eocene-age fluvial systems, consistent with a fluvial clastic response to Paleogene warming. We suggest that deep-marine sedimentary basins preserve clastic records of early Eocene climatic extremes. We also suggest that in addition to control by eustasy and tectonism, climate-driven increases in sediment supply (e.g., drainage integration, global rainfall, denudation) may significantly contribute to the global distribution and volume of coarse-grained deep-marine deposition despite high sea level.

Interaction of RNA polymerase II fork loop 2 with downstream non-template DNA regulates transcription elongation.

Fork loop 2 is a small semiconservative segment of the larger fork domain in the second largest Rpb2 subunit of RNA polymerase II (Pol II). This flexible loop, juxtaposed at the leading edge of transcription bubble, has been proposed to participate in DNA strand separation, translocation along DNA, and NTP loading to Pol II during elongation. Here we show that the Rpb2 mutant carrying a deletion of the flexible part of the loop is not lethal in yeast. The mutation exhibits no defects in DNA melting and translocation in vitro but confers a moderate decrease of the catalytic activity of the enzyme caused by the impaired sequestration of the NTP substrate in the active center prior to catalysis. In the structural model of the Pol II elongation complex, fork loop 2 directly interacts with an unpaired DNA residue in the non-template DNA strand one nucleotide ahead from the active center (the i+2 position). We showed that elimination of this putative interaction by replacement of the i+2 residue with an abasic site inhibits Pol II activity to the same degree as the deletion of fork loop 2. This replacement has no detectable effect on the activity of the mutant enzyme. We provide direct evidence that interaction of fork loop 2 with the non-template DNA strand facilitates NTP sequestration through interaction with the adjacent segment of the fork domain involved in the active center of Pol II.

Navigating mental health challenges in graduate school.

Many graduate students experience mental health struggles that lead them to question their place in academia. Two scientists who experienced extreme lows in graduate school reflect on what helped them during their low points, and suggest strategies for everyone to contribute to mentally healthier workplaces in academia.

Translocation by multi-subunit RNA polymerases.

DNA template and RNA/DNA hybrid movement through RNA polymerase (RNAP) is referred to as "translocation". Because nucleic acid movement is coupled to NTP loading, pyrophosphate release, and conformational changes, the precise ordering of events during bond addition is consequential. Moreover, based on several lines of experimental evidence, translocation, pyrophosphate release or an associated conformational change may determine the transcription elongation rate. In this review we discuss various models of translocation, the data supporting the hypothesis that translocation rate determines transcription elongation rate and also data that may be inconsistent with this point of view. A model of the nucleotide addition cycle accommodating available experimental data is proposed. On the basis of this model, the molecular mechanisms regulating translocation and potential routes for NTP entry are discussed.

Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase.

Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant beta R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge alpha-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in beta R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site "latch" assembly that includes a key trigger helix residue Tt beta' H1242 and highly conserved active site residues beta E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.

Early Evolution of Transcription Systems and Divergence of Archaea and Bacteria.

DNA template-dependent multi-subunit RNA polymerases (RNAPs) found in all three domains of life and some viruses are of the two-double-Ψ-ß-barrel (DPBB) type. The 2-DPBB protein format is also found in some RNA template-dependent RNAPs and a major replicative DNA template-dependent DNA polymerase (DNAP) from Archaea (PolD). The 2-DPBB family of RNAPs and DNAPs probably evolved prior to the last universal common cellular ancestor (LUCA). Archaeal Transcription Factor B (TFB) and bacterial σ factors include homologous strings of helix-turn-helix units. The consequences of TFB-σ homology are discussed in terms of the evolution of archaeal and bacterial core promoters. Domain-specific DPBB loop inserts functionally connect general transcription factors to the RNAP active site. Archaea appear to be more similar to LUCA than Bacteria. Evolution of bacterial σ factors from TFB appears to have driven divergence of Bacteria from Archaea, splitting the prokaryotic domains.

RNA polymerase II with open and closed trigger loops: active site dynamics and nucleic acid translocation.

RNA polymerase II is the central eukaryotic enzyme in transcription from DNA to RNA. The dynamics of RNA polymerase II is described from molecular-dynamics simulations started from two crystal structures with open and closed trigger loop (TL) forms. Dynamic transitions between neutral and forward translocated states were observed, especially for the downstream DNA duplex. Dynamic rearrangements were also seen in the active site environment, including conformations in which the active site nucleotide assumed a possibly precatalytic conformation in close proximity to the terminal 3'-hydroxyl of the nascent RNA. Because nucleic acid translocation was observed primarily in the simulations with an open TL structure, whereas close approach of the active site nucleotide to the terminal RNA ribose predominantly occurred in the closed TL structure, a modified Brownian ratchet mechanism is proposed whereby thermally driven translocation is only possible with an open TL, and fidelity control and catalysis require TL closing.

Minimal promoter systems reveal the importance of conserved residues in the B-finger of human transcription factor IIB.

The "B-finger" of transcription factor IIB (TFIIB) is highly conserved and believed to play a role in the initiation process. We performed alanine substitutions across the B-finger of human TFIIB, made change-of-charge mutations in selected residues, and substituted the B-finger sequence from other organisms. Mutant proteins were examined in two minimal promoter systems (containing only RNA polymerase II, TATA-binding protein, and TFIIB) and in a complex system, using TFIIB-immunodepleted HeLa cell nuclear extract (NE). Mutations in conserved residues located on the sides of the B-finger had the greatest effect on activity in both minimal promoter systems, with mutations in residues Glu-51 and Arg-66 eliminating activity. The double change-of-charge mutant (E51R:R66E) did not show activity in either minimal promoter system. Mutations in the nonconserved residues at the tip of the B-finger did not significantly affect activity. However, all of the mutations in the B-finger showed at least 25% activity in the HeLa cell NE. Chimeric proteins, containing B-finger sequences from species with conserved residues on the side of the B-finger, showed wild-type activity in a minimal promoter system and in the HeLa cell NE. However, chimeric proteins whose sequence showed divergence on the sides of the B-finger had reduced activity. Transcription factor IIF (TFIIF) partially restored activity of the inactive mutants in the minimal promoter system, suggesting that TFIIF in HeLa cell NE helps to rescue the inactive mutations by interacting with either the B-finger or another component of the initiation complex that is influenced by the B-finger.

RNA polymerase II flexibility during translocation from normal mode analysis.

The structural dynamics in eukaryotic RNA polymerase II (RNAPII) is described from computational normal mode analysis based on a series of crystal structures of pre- and post-translocated states with open and closed trigger loops. Conserved modes are identified that involve translocation of the nucleic acid complex coupled to motions of the enzyme, in particular in the clamp and jaw domains of RNAPII. A combination of these modes is hypothesized to be involved during active transcription. The NMA modes indicate furthermore that downstream DNA translocation may occur separately from DNA:RNA hybrid translocation. A comparison of the modes between different states of RNAPII suggests that productive translocation requires an open trigger loop and is inhibited by the presence of an NTP in the active site. This conclusion is also supported by a comparison of the overall flexibility in terms of root mean square fluctuations.

Site-directed mutagenesis, purification and assay of Saccharomyces cerevisiae RNA polymerase II.

In order to analyze the structure-function of multi-subunit RNA polymerases (RNAPs), it is necessary to make site-directed mutations in key residues. Because Saccharomyces cerevisiae RNAP II is isolated as a 12 subunit enzyme that has not been amenable to in vitro reconstitution, making site-directed mutations in a particular subunit presents technical issues. In this work, we demonstrate a method to generate and purify site-directed mutants in the second largest (Rpb2) RNAP II subunit from yeast, using a tandem affinity purification tag. Mutants are analyzed for growth defects in vivo and for defects in transcriptional elongation in vitro. We show that Rpb2 R512A/C located just C-terminal to fork loop 2 (Rpb2 500-511) has transcriptional defects that are distinct from surrounding fork loop 2 region mutants. Rpb2 E529A/D replacements are faster and E529Q is slower than wild type RNAP II in elongation. E529 appears to form an ion pair with K987, an essential active site residue. Mutations are also analyzed within the active site region indicating key residues for catalysis and the importance of a Rpb2 R983-E1028 ion pair. Rpb2 R983Q and E1028Q are defective in escape from a transcriptional stall.