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.