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.

Applications in micro/nanoelectromechanical systems generally require low adhesion and friction values between two materials of interest. By alteration of the material combinations and surface roughness, including nanopatterning, adhesion and friction can be tailored to meet a specific requirement. Surfaces found in nature, such as hydrophobic lotus leaves, provide a good example of this optimization. Recent models of hydrophobic leaf surfaces show a correlation between roughness and hydrophobicity, which can be mimicked by the presence of nanopatterned asperities on a polymer surface. In addition, by introducing nanopatterns on the polymer surface, the real area of contact decreases when another surface comes into contact with the patterned surface, which reduces adhesion and friction. This study explores the effect of nanopatterning on hydrophobicity, adhesion, and friction for two different hydrophilic polymers, poly(methyl methacrylate) (PMMA) and polyurethane acrylate (MINS), with two types of patterned asperities, low aspect ratio and high aspect ratio, investigated by use of an atomic/friction force microscope (AFM/FFM). In addition to the polymers, a hydrophobic coating was deposited on the surface of the patterned PMMA to study the effect of roughness on the contact angle, along with adhesion and friction. Relative contribution due to change in contact angle and real area of contact are explored. Scale dependence on adhesion and friction was also studied using AFM tips of various radii. Since applications of these surfaces will require operation in varying environments, the effect of relative humidity is investigated.

The products of the yeast CDC73 and PAF1 genes were originally identified as RNA polymerase II-associated proteins. Paf1p is a nuclear protein important for cell growth and transcriptional regulation of a subset of yeast genes. In this study we demonstrate that the product of CDC73 is a nuclear protein that interacts directly with purified RNA polymerase II in vitro. Deletion of CDC73 confers a temperature-sensitive phenotype. Combination of the cdc73 mutation with the more severe paf1 mutation does not result in an enhanced phenotype, indicating that the two proteins may function in the same cellular processes. To determine the relationship between Cdc73p and Paf1p and the recently described holoenzyme form of RNA polymerase II, we created yeast strains containing glutathione S-transferase ( A minimal set of transcription factors (RNA polymerase II plus TATA-binding protein [TBP], TFIIB, TFIIE, TFIIF, and TFIIH) are necessary for mRNA promoter-specific transcription initiation in vitro (for reviews, see references 7 and 12). Regulated transcription requires, in addition to these basal factors, many accessory proteins responsible for conveying regulatory signals to the general transcriptional machinery (68). There are at least two different classes of accessory factors that have been well characterized. One class includes the TBPassociated factors (TAFs) (for a review, see reference 58). In vitro reconstitution experiments strongly implicate the TAFs in the process of transcriptional activation (10). Another class of accessory factors exists in the mediator complex associated with the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II (for a review, see reference 33). In the yeast Saccharomyces cerevisiae, the mediator can associate with RNA polymerase II and several general initiation factors to form a large protein complex termed the holoenzyme (30, 32). Most components of the holoenzyme, including the Srbps, Gal11p, Sin4p, Rgr1p, and Swi/Snfps, were originally identified by mutations that caused transcriptional alterations in yeast (24,27,34,39,43,53,64). Although mutations in some of these gene products affect the expression of only subsets of yeast genes, an analysis of temperature-sensitive mutations of SRB4 and SRB6 revealed transcription defects at all class II promoters assayed (57). Mammalian RNA polymerase II-containing complexes that include Srbp homologs, and many of the general transcription factors as well as DNA-repair factors, have recently been described (36,42).The reported complex forms of RNA polymerase II vary widely in terms of composition. In particular, some of the general initiation factors (TBP, TFIIE, and TFIIH) are present in some complexes but not others (30,32,36,42). In addition, some of the factors, including the Srbps and Gal11p, can be found in dissociable subcomplexes (30,34). Although it is probable that some of these differences reflect the different purification protocols used to isolate these extremely large complexes from widely differing cell type...

Each cycle of transcription appears to be associated with the reversible phosphorylation of the repetitive COOH-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit. The dephosphorylation of RNAP II by CTD phosphatase, therefore, plays an important role in the transcription cycle. The following studies characterize the activity of HeLa cell CTD phosphatase with a special emphasis on the regulation of CTD phosphatase activity. Results presented here suggest that RNAP II contains a docking site for CTD phosphatase that is essential in the dephosphorylation reaction and is distinct from the CTD. This is supported by the observations that (a) phosphorylated recombinant CTD is not a substrate for CTD phosphatase, (b) RNAP IIB, which lacks the CTD, and RNAP IIA are competitive inhibitors of CTD phosphatase and (c) CTD phosphatase can form a stable complex with RNAP II. To test the possibility that the general transcription factors may be involved in the regulation of CTD phosphatase, CTD phosphatase activity was examined in the presence of recombinant or highly purified general transcription factors. TFIIF stimulates CTD phosphatase activity 5-fold. The RAP74 subunit of TFIIF alone contained the stimulatory activity and the minimal region sufficient for stimulation corresponds to COOH-terminal residues 358-517. TFIIB inhibits the stimulatory activity of TFIIF but has no effect on CTD phosphatase activity in the absence of TFIIF. The potential importance of the docking site on RNAP II and the effect of TFIIF and TFIIB in regulating the dephosphorylation of RNAP II at specific times in the transcription cycle are discussed.

Regulated transcription initiation requires, in addition to RNA polymerase II and the general transcription factors, accessory factors termed mediators or adapters. We have used affinity chromatography to identify a collection of factors that associate with Saccharomyces cerevisiae RNA polymerase II (P. A. Wade, W. Werel, R. C. Fentzke, N. E. Thompson, J. F. Leykam, R. R. Burgess, J. A. Jaehning, and Z. F. Burton, submitted for publication). Here we report identification and characterization of a gene encoding one of these factors, PAF1 (for RNA polymerase-associated factor 1). PAF1 encodes a novel, highly charged protein of 445 amino acids. Disruption of PAF1 in S. cerevisiae leads to pleiotropic phenotypic traits, including slow growth, temperature sensitivity, and abnormal cell morphology. Consistent with a possible role in transcription, Paf1p is localized to the nucleus. By comparing the abundances of many yeast transcripts in isogenic wild-type and paf1 mutant strains, we have identified genes whose expression is affected by PAF1. In particular, disruption of PAF1 decreases the induction of the galactose-regulated genes three- to fivefold. In contrast, the transcript level of MAK16, an essential gene involved in cell cycle regulation, is greatly increased in the paf1 mutant strain. Paf1p may therefore be required for both positive and negative regulation of subsets of yeast genes. Like Paf1p, the GAL11 gene product is found associated with RNA polymerase II and is required for regulated expression of many yeast genes including those controlled by galactose. We have found that a gal11 paf1 double mutant has a much more severe growth defect than either of the single mutants, indicating that these two proteins may function in parallel pathways to communicate signals from regulatory factors to RNA polymerase II.

We advocate for a tRNA- rather than an mRNA-centric model for evolution of the genetic code. The mechanism for evolution of cloverleaf tRNA provides a root sequence for radiation of tRNAs and suggests a simplified understanding of code evolution. To analyze code sectoring, rooted tRNAomes were compared for several archaeal and one bacterial species. Rooting of tRNAome trees reveals conserved structures, indicating how the code was shaped during evolution and suggesting a model for evolution of a LUCA tRNAome tree. We propose the polyglycine hypothesis that the initial product of the genetic code may have been short chain polyglycine to stabilize protocells. In order to describe how anticodons were allotted in evolution, the sectoring-degeneracy hypothesis is proposed. Based on sectoring, a simple stepwise model is developed, in which the code sectors from a 1→4→8→∼16 letter code. At initial stages of code evolution, we posit strong positive selection for wobble base ambiguity, supporting convergence to 4-codon sectors and ∼16 letters. In a later stage, ∼5–6 letters, including stops, were added through innovating at the anticodon wobble position. In archaea and bacteria, tRNA wobble adenine is negatively selected, shrinking the maximum size of the primordial genetic code to 48 anticodons. Because 64 codons are recognized in mRNA, tRNA-mRNA coevolution requires tRNA wobble position ambiguity leading to degeneracy of the code.

We report a "running start, two-bond" protocol to analyze elongation by human RNA polymerase II (RNAP II). In this procedure, the running start allowed us to measure rapid rates of elongation and provided detailed insight into the RNAP II mechanism. Formation of two bonds was tracked to ensure that at least one translocation event was analyzed. By using this method, RNAP II is stalled briefly at a defined template position before restoring the next NTP. Significantly, slow reaction steps are identified both before and after phosphodiester bond synthesis, and both of these steps can be highly dependent on the next templated NTP. The initial and final NTP-driven events, however, are not identical, because the slow step after chemistry, which includes translocation and pyrophosphate release, is regulated differently by elongation factors hepatitis ␦ antigen and transcription factor IIF. Because recovery from a stall and the processive transition from one bond to the next can be highly NTP-dependent, we conclude that translocation can be driven by the incoming substrate NTP, a model fully consistent with the RNAP II elongation complex structure.Pre-steady state kinetic analysis allows the progress of an enzymatic reaction to be tracked in real time (1, 2), and coupling enzyme functional dynamics to the structure provides the clearest insight into the mechanism. In this paper, we compare the first transient state kinetic studies of human (Homo sapiens) RNAP II 1 to the x-ray structure of the yeast (Saccharomyces cerevisiae) RNAP II elongation complex (EC) (3). These studies give new insight into the RNAP II mechanism and demonstrate the feasibility of a detailed kinetic study of a highly regulated enzyme that is at the hub of gene control in human cells.There is increasing recognition that transcriptional elongation is highly regulated in eukaryotes (4 -8). As an example, hepatitis ␦ antigen (HDAg) strongly stimulates RNAP II elongation in vitro (6, 9). HDAg is the sole gene product of the small RNA genome of hepatitis ␦ virus, which is maintained as a satellite particle by hepatitis B virus. The role of HDAg in elongation may be clinically significant because hepatitis ␦ virus often complicates severe and chronic presentations of human hepatitis B virus infection. The general cellular transcription factor IIF (TFIIF) has been shown to stimulate RNAP II elongation 5-10-fold in vitro, by suppressing transcriptional pausing (10 -16). The role of TFIIF in elongation may be of particular importance during the promoter escape phase of the transcription cycle (17, 18). Here viral HDAg and cellular TFIIF are used as probes of H. sapiens RNAP II elongation.In this work, we use rapid quench kinetics to demonstrate critical NTP-dependent steps during RNA synthesis. First, we analyzed recovery from a stall at a defined template position, in the presence of TFIIF or HDAg. During stall recovery, two fractions of EC were clearly observed on the active pathway, and most significantly, these ECs had different requirements for bindin...

Pathways of standard genetic code evolution remain conserved and apparent, particularly upon analysis of aminoacyl-tRNA synthetase (aaRS) lineages. Despite having incompatible active site folds, class I and class II aaRS are homologs by sequence. Specifically, structural class IA aaRS enzymes derive from class IIA aaRS enzymes by in-frame extension of the protein N-terminus and by an alternate fold nucleated by the N-terminal extension. The divergence of aaRS enzymes in the class I and class II clades was analyzed using the Phyre2 protein fold recognition server. The class I aaRS radiated from the class IA enzymes, and the class II aaRS radiated from the class IIA enzymes. The radiations of aaRS enzymes bolster the coevolution theory for evolution of the amino acids, tRNAomes, the genetic code, and aaRS enzymes and support a tRNA anticodon-centric perspective. We posit that second- and third-position tRNA anticodon sequence preference (C>(U~G)>A) powerfully selected the sectoring pathway for the code. GlyRS-IIA appears to have been the primordial aaRS from which all aaRS enzymes evolved, and glycine appears to have been the primordial amino acid around which the genetic code evolved.