Transcription start site scanning and the requirement for ATP during transcription initiation by RNA polymerase II

Saccharomyces cerevisiae RNA polymerase (Pol) II locates transcription start sites (TSS) at TATA-containing promoters by scanning sequences downstream from the site of preinitiation complex formation, a process that involves the translocation of downstream promoter DNA toward Pol II. To investigate a potential role of yeast Pol II transcription in TSS scanning, HIS4 promoter derivatives were generated that limited transcripts in the 30-bp scanned region to two nucleotides in length. Although we found that TSS scanning does not require RNA synthesis, our results revealed that transcription in the purified yeast basal system is largely ATP-independent despite a requirement for the TFIIH DNA translocase subunit Ssl2. This result is rationalized by our finding that, although they are poorer substrates, UTP and GTP can also be utilized by Ssl2. ATPγS is a strong inhibitor of rNTP-fueled translocation, and high concentrations of ATPγS make transcription completely dependent on added dATP. Limiting Pol II function with low ATP concentrations shifted the TSS position downstream. Combined with prior work, our results show that Pol II transcription plays an important role in TSS selection but is not required for the scanning reaction

Site-directed mutagenesis of the proposed catalytic amino acids of the Sindbis virus capsid protein autoprotease

The structural proteins of Sindbis virus are translated as a polyprotein precursor that is cleaved upon translation. The capsid protein is postulated to be a serine protease that releases itself from the N terminus of the nascent polyprotein by autoproteolysis. We have tested the importance in autoproteolysis of His-141, Asp-147, and Ser-215, previously postulated to form the catalytic triad of the protease, and of Asp-163. Several site-specific mutations were constructed at each of these positions, and the release of the capsid protein during translation in a cell-free system was examined. Because proteolysis occurs in cis during translation, the kinetics of release cannot be determined in this system, but the extent of proteolysis can be ascertained. Ser-215 appears to be the catalytic serine of the proteinase. Cys or Thr could substitute inefficiently for Ser-215, but substitution with Ala or Ile led to complete loss of activity. His-141 was also important for proteolysis. Substitution with Ala or Pro led to total loss of activity. Surprisingly, substitution with Arg resulted in complete proteolysis in vitro. Changes at the two Asp residues resulted in complete proteolysis of the substrate in vitro. All mutations that resulted in at least partial cleavage in vitro were incorporated into a full-length clone of Sindbis virus and an attempt was made to recover mutant virus. All changes tested were lethal for the virus except Asp-163 to Asn. Thus, production of infectious virus is either a more sensitive measure of the catalytic rate than the extent of in vitro cleavage, or these residues have necessary functions in addition to their possible role in proteolysis

The Impact of Driver Cell Phone Use on Accidents

Cell phone use is increasing worldwide, leading to a concern that cell phone use while driving increases accidents. We develop a new approach for estimating the relationship between cell phone use while driving and accidents, based on new survey data. We test for selection effects, such as whether drivers who use cell phones are inherently less safe drivers, even when not on the phone. The paper has two key findings. First, the impact of cell phone use on accidents varies across the population. This result implies that previous estimates of the impact of cell phone use on risk for the population, based on accident-only samples, may therefore be overstated by 36%. Second, once we correct for endogeneity, there is no significant effect of hands-free or hand-held cell phone use on accidents.cellular telephones and driving, safety regulation, selection effects

The Impact of Driver Cell Phone Use on Accidents

Cell phone use is increasing worldwide, leading to a concern that cell phone use while driving increases accidents. Several countries, three states and Washington, D.C. have banned the use of hand-held cell phones while driving. In this paper, we develop a new approach for estimating the relationship between cell phone use while driving and accidents. Our approach is the first to allow for the direct estimation of the impact of a cell phone ban while driving. It is based on new survey data from over 7,000 individuals. This paper differs from previous research in two significant ways: first, we use a larger sample of individual-level data; and second, we test for selection effects, such as whether drivers who use cell phones are inherently less safe drivers, even when not on the phone. The paper has two key findings. First, the impact of cell phone use on accidents varies across the population. This result implies that previous estimates of the impact of cell phone use on risk for the population, based on accident-only samples, may be overstated by about one-third. Second, once we correct for endogeneity, there is no significant effect of hands-free or hand-held cell phone use on accidents.

The Impact of Driver Cell Phone Use on Accidents

Cell phone use is increasing worldwide, leading to a concern that cell phone use while driving increases accidents. Several countries, three states and Washington, D.C. have banned the use of hand-held cell phones while driving. In this paper, we develop a new approach for estimating the relationship between cell phone use while driving and accidents. Our approach is the first to allow for the direct estimation of the impact of a cell phone ban while driving. It is based on new survey data from over 7,000 individuals. This paper differs from previous research in two significant ways: first, we use a larger sample of individual-level data; and second, we test for selection effects, such as whether drivers who use cell phones are inherently less safe drivers, even when not on the phone. The paper has two key findings. First, the impact of cell phone use on accidents varies across the population. This result implies that previous estimates of the impact of cell phone use on risk for the population, based on accident-only samples, may be overstated by about one-third. Second, once we correct for endogeneity, there is no significant effect of hands-free or hand-held cell phone use on accidents.

Periodic Orbits for a Discontinuous Vector Field Arising from a Conceptual Model of Glacial Cycles

Conceptual climate models provide an approach to understanding climate processes through a mathematical analysis of an approximation to reality. Recently, these models have also provided interesting examples of nonsmooth dynamical systems. Here we discuss a conceptual model of glacial cycles consisting of a system of three ordinary differential equations defining a discontinuous vector field. We show that this system has a large periodic orbit crossing the discontinuity boundary. This orbit can be interpreted as an intrinsic cycling of the Earth's climate giving rise to alternating glaciations and deglaciations

Mapping of RNA- temperature-sensitive mutants of Sindbis virus: assignment of complementation groups A, B, and G to nonstructural proteins

Four complementation groups of temperature-sensitive (ts) mutants of Sindbis virus that fail to make RNA at the nonpermissive temperature are known, and we have previously shown that group F mutants have defects in nsP4. Here we map representatives of groups A, B, and G. Restriction fragments from a full-length clone of Sindbis virus, Toto1101, were replaced with the corresponding fragments from the various mutants. These hybrid plasmids were transcribed in vitro by SP6 RNA polymerase to produce infectious RNA transcripts, and the virus recovered was tested for temperature sensitivity. After each lesion was mapped to a specific region, cDNA clones of both mutants and revertants were sequenced in order to determine the precise nucleotide change responsible for each mutation. Synthesis of viral RNA and complementation by rescued mutants were also examined in order to study the phenotype of each mutation in a uniform genetic background. The single mutant of group B, ts11, had a defect in nsP1 (Ala-348 to Thr). All of the group A and group G mutants examined had lesions in nsP2 (Ala-517 to Thr in ts17, Cys-304 to Tyr in ts21, and Gly-736 to Ser in ts24 for three group A mutants, and Phe-509 to Leu in ts18 and Asp-522 to Asn in ts7 for two group G mutants). In addition, ts7 had a change in nsP3 (Phe-312 to Ser) which also rendered the virus temperature sensitive and RNA-. Thus, changes in any of the four nonstructural proteins can lead to failure to synthesize RNA at a nonpermissive temperature, indicating that all four are involved in RNA synthesis. From the results presented here and from previous results, several of the activities of the nonstructural proteins can be deduced. It appears that nsP1 may be involved in the initiation of minus-strand RNA synthesis. nsP2 appears to be involved in the initiation of 26S RNA synthesis, and in addition it appears to be a protease that cleaves the nonstructural polyprotein precursors. It may also be involved in shutoff of minus-strand RNA synthesis. nsP4 appears to function as the viral polymerase or elongation factor. The functions of nsP3 are as yet unresolved

The Effect of Cooperative Learning on Test Performance

The purpose of this study was to determine if cooperative learning in the form of study games could have an effect on student test performance. The subjects were 96 eighth grade students at a suburban New York State school. The 96 students were divided into 4 classes of 24 each. The course that the students were enrolled in was physical science, which was a mandatory course at this particular school. The cooperative learning technique of utilizing games as a means of study was developed by Robert E. Slavin, at Johns Hopkins University. The particular series and steps of the games in this study were a spin-off of Slavin\u27s work with a few different steps added. The games consisted of the teacher developing a list of questions and answers which were later transferred to flashcards by the students. The questions and answers pertained to the topic being studied and were the basis of the exam which followed. The students were divided into teams within the classroom and competed for points which were scored as a result of correctly answering the flashcard of the opposing team. For the purpose of analysis, an independent t-test was used to determine if there was a significant difference between the mean test scores of the students who played the game (treatment group), and the students who did not play the game (non treatment group). The treatment group and the non treatment group exchanged roles after 10 weeks, which consisted of taking 2 exams, where the treatment group became the non treatment group and the non treatment group became the treatment group. Statistical analysis was performed on each exam taken by the students during a total of 20 weeks. The results showed, in every exam, that the students who had participated in the cooperative learning experience obtained higher test scores than did those students not participating. On average the treatment group\u27s test score mean was 5 points higher than the non treatment group\u27s test score mean. In one case the treatment group\u27s test score mean was 15 points higher than the non treatment group\u27s test score mean