Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise.

Transcription initiation is highly regulated by promoter sequence, transcription factors, and ligands. All known transcription inhibitors, an important class of antibiotics, act in initiation. To understand regulation and inhibition, the biophysical mechanisms of formation and stabilization of the "open" promoter complex (OC), of synthesis of a short RNA-DNA hybrid upon nucleotide addition, and of escape of RNA polymerase (RNAP) from the promoter must be understood. We previously found that RNAP forms three different OC with λPR promoter DNA. The 37 °C RNAP-λPR OC (RPO) is very stable. At lower temperatures, RPO is less stable and in equilibrium with an intermediate OC (I3). Here, we report step-by-step rapid quench-flow kinetic data for initiation and growth of the RNA-DNA hybrid at 25 and 37 °C that yield rate constants for each step of productive nucleotide addition. Analyzed together, with previously published data at 19 °C, our results reveal that I3 and not RPO is the productive initiation complex at all temperatures. From the strong variations of rate constants and activation energies and entropies for individual steps of hybrid extension, we deduce that contacts of RNAP with the bubble strands are disrupted stepwise as the hybrid grows and translocates. Stepwise disruption of RNAP-strand contacts is accompanied by stepwise bubble collapse, base stacking, and duplex formation, as the hybrid extends to a 9-mer prior to disruption of upstream DNA-RNAP contacts and escape of RNAP from the promoter.

RNA Polymerase: Step-by-Step Kinetics and Mechanism of Transcription Initiation.

To determine the step-by-step kinetics and mechanism of transcription initiation and escape by E. coli RNA polymerase from the λPR promoter, we quantify the accumulation and decay of transient short RNA intermediates on the pathway to promoter escape and full-length (FL) RNA synthesis over a wide range of NTP concentrations by rapid-quench mixing and phosphorimager analysis of gel separations. Experiments are performed at 19 °C, where almost all short RNAs detected are intermediates in FL-RNA synthesis by productive complexes or end-products in nonproductive (stalled) initiation complexes and not from abortive initiation. Analysis of productive-initiation kinetic data yields composite second-order rate constants for all steps of NTP binding and hybrid extension up to the escape point (11-mer). The largest of these rate constants is for incorporation of UTP into the dinucleotide pppApU in a step which does not involve DNA opening or translocation. Subsequent steps, each of which begins with reversible translocation and DNA opening, are slower with rate constants that vary more than 10-fold, interpreted as effects of translocation stress on the translocation equilibrium constant. Rate constants for synthesis of 4- and 5-mer, 7-mer to 9-mer, and 11-mer are particularly small, indicating that RNAP-promoter interactions are disrupted in these steps. These reductions in rate constants are consistent with the previously determined ∼9 kcal cost of escape from λPR. Structural modeling and previous results indicate that the three groups of small rate constants correspond to sequential disruption of in-cleft, -10, and -35 interactions. Parallels to escape by T7 RNAP are discussed.

Assessing effort: differentiating performance and symptom validity.

The current study aimed to clarify the relationship among the constructs involved in neuropsychological assessment, including cognitive performance, symptom self-report, performance validity, and symptom validity. Participants consisted of 120 consecutively evaluated individuals from a veteran's hospital with mixed referral sources. Measures included the Wechsler Adult Intelligence Scale-Fourth Edition Full Scale IQ (WAIS-IV FSIQ), California Verbal Learning Test-Second Edition (CVLT-II), Trail Making Test Part B (TMT-B), Test of Memory Malingering (TOMM), Medical Symptom Validity Test (MSVT), WAIS-IV Reliable Digit Span (RDS), Post-traumatic Check List-Military Version (PCL-M), MMPI-2 F scale, MMPI-2 Symptom Validity Scale (FBS), MMPI-2 Response Bias Scale (RBS), and the Postconcussive Symptom Questionnaire (PCSQ). Six different models were tested using confirmatory factor analysis (CFA) to determine the factor model describing the relationships between cognitive performance, symptom self-report, performance validity, and symptom validity. The strongest and most parsimonious model was a three-factor model in which cognitive performance, performance validity, and self-reported symptoms (including both standard and symptom validity measures) were separate factors. The findings suggest failure in one validity domain does not necessarily invalidate the other domain. Thus, performance validity and symptom validity should be evaluated separately.

Seasonal bioaccumulation of organohalogens in tigerfish, Hydrocynus vittatus Castelnau, from Lake Pongolapoort, South Africa.

The study determined current exposure of tigerfish in Lake Pongolaqpoort to organohalogens. Levels of DDT, PCB, HCB, HCH, PBDE and CHLs were measured in tigerfish muscle on a seasonal basis. Historical use of DDT was reflected in the bioaccumulation patterns (5,400-6,000 ng/g lipid) as well as current use of HCBs (7.7-15.7 ng/g lipid) in the agricultural areas. External factors, i.e. increased flow did not play a role in organic pollutant exposure. Levels are a function of the lipid content of the muscle tissue, i.e. 3.8% during low and 9% during high flow, implying that organohalogen exposure remains fairly constant throughout the year.

The utility of the post-concussive symptom questionnaire.

The Post-concussive Symptom Questionnaire (PCSQ) and its short forms were evaluated to determine their utility in measuring symptom validity as brief self-report measures in 112 individuals referred for a neuropsychological evaluation. First, the relationships between the PCSQ forms and measures of cognitive performance (Wechsler Adult Intelligence Scale-Fourth Edition Full-Scale IQ, California Verbal Learning Test-Second Edition Trials 1-5 Total T-score, Trails B, FAS), general distress (Minnesota Multiphasic Personality Inventory [MMPI]-2 M8), and self-report symptom validity (MMPI-2 FBS Symptom Validity Scale [FBS] and Response Bias Scale [RBS]) were investigated to determine construct validity. Measures of self-report symptom validity explained the greatest amount of variance. Second, receiver operating characteristics curve analyses were conducted to determine the predictive value of the PCSQ forms in detecting over reporting on the FBS and the RBS in addition to establishing optimal cutoff scores. On the basis of the proposed cutoff scores, sensitivity, specificity, positive predictive power, negative predictive power, and hit rates were calculated.

Test-retest reliability of the Traumatic Brain Injury Screening Instrument.

The Traumatic Brain Injury Screening Instrument (TBISI) was implemented in Veterans Affairs medical facilities in an attempt to identify Operation Enduring Freedom and Operation Iraqi Freedom (OEF/OIF) veterans with possible mild TBI. Despite its widespread implementation, the reliability and validity of the screening tool has not yet been established. The current study reports preliminary findings on the test-retest reliability of the TBISI in 44 OEF/OIF veterans referred for neuropsychological evaluation following a positive TBI screen. Results suggest overall poor test-retest reliability of the TBI screening tool with regard to type of event, injuries sustained, and resulting sequelae. These findings underscore the importance of further investigation of its psychometric properties to promote accurate identification and referral of veterans with possible TBI.

Gender differences in lateralized semantic priming.

Previous literature suggests that women evidence more bilateral cerebral organization, particularly in language processing, whereas men show greater left hemisphere dominance for language. This study examined the magnitude of these gender differences in a lateralized lexical decision task and the implications of such differences to semantic processing and cerebral organization. As predicted, women, as compared to men, recruited greater bilateral hemispheric resources, as evidenced by greater contralateral hemispheric priming. Spatial skills predicted less priming in women, but not in men. Implications for laterality research in aging populations as well as future directions are discussed.

Validity of demographically corrected norms for the WAIS-III.

The diagnostic validity of new demographically corrected WAIS-III norms was investigated using a sample of 100 patients with traumatic brain injury (TBI) and a matched control group from the standardization sample. Demographically corrected norms were compared to traditional age-corrected norms. Although education accounted for incremental variance in WAIS-III factor scores in patients with TBI, above and beyond the effects of injury severity, the demographically corrected norms did not yield statistically different diagnostic classification of individuals with moderate-severe TBI than the traditional norms. In participants with relatively low levels of educational attainment, sensitivity to length of coma was less for demographically corrected norms then for traditional age-corrected norms. Nevertheless, when using a discrepancy between Verbal Comprehension and Processing speed, diagnostic accuracy rates were again similar for both sets of norms. It is concluded that the demographically corrected WAIS-III norms do not offer a clear advantage or disadvantage compared to traditional age-corrected norms in the assessment of patients with TBI who are Caucasian and who have at least a middle school level of education.