Videotaped interviews as a medium to enhance cross-cultural programme evaluation.

Evaluation is a required component of interventions. Written data are the predominant source. However, video recording is used in many applications to evaluate a range of encounters and practices. We report assessment of the role of videotaped interviews in programme evaluation. Interviews using a consistent script of open-ended questions were recorded during evaluation of an international child-health promotion programme in Uganda by individuals with basic training and equipment. Participants were a convenience sample of programme team members (six school teachers, and six Ugandan and 12 Canadian health-care trainees) who had completed the annual written evaluation questionnaire. Evaluators reviewed each participant's videotaped interview and questionnaire, content coded the responses against a criterion-based check list, documented how many times factual information was contributed on each question and compared the data. Videos were also assessed for strong positive or negative emotion. Videotaped interviews provided more comprehensive responses than written questionnaires, and were more accurate where mis-comprehension of question meaning occurred. The video interview, unlike the written questionnaire, allowed rephrasing for clarification. The video interview medium enhanced programme evaluation by providing more facts, greater insight into the effects of the interventions and clearer direction for future activity. Hence, video-recorded feedback has great potential value in applied research for comprehensive programme evaluation.

Coupling sigma factor conformation to RNA polymerase reorganisation for DNA melting.

ATP-driven remodelling of initial RNA polymerase (RNAP) promoter complexes occurs as a major post recruitment strategy used to control gene expression. Using a model-enhancer-dependent bacterial system (sigma54-RNAP, Esigma54) and a slowly hydrolysed ATP analogue (ATPgammaS), we provide evidence for a nucleotide-dependent temporal pathway leading to DNA melting involving a small set of sigma54-DNA conformational states. We demonstrate that the ATP hydrolysis-dependent remodelling of Esigma54 occurs in at least two distinct temporal steps. The first detected remodelling phase results in changes in the interactions between the promoter specificity sigma54 factor and the promoter DNA. The second detected remodelling phase causes changes in the relationship between the promoter DNA and the core RNAP catalytic beta/beta' subunits, correlating with the loading of template DNA into the catalytic cleft of RNAP. It would appear that, for Esigma54 promoters, loading of template DNA within the catalytic cleft of RNAP is dependent on fast ATP hydrolysis steps that trigger changes in the beta' jaw domain, thereby allowing acquisition of the open complex status.

Nucleotide-dependent triggering of RNA polymerase-DNA interactions by an AAA regulator of transcription.

Enhancer-dependent activator proteins, which act upon the bacterial RNA polymerase containing the sigma54 promoter specificity factor, belong to the AAA superfamily of ATPases. Activator-sigma54 contact is required for the sigma54-RNAP to isomerize and engage the DNA template for transcription. How ATP hydrolysis is used to trigger changes in sigma54-RNA polymerase and promoter DNA that lead to DNA opening is poorly understood. Here, band shift and footprinting assays were used to investigate the DNA binding activities of sigma54 and sigma54-RNA polymerase in the presence of the activator protein PspF bound to poorly hydrolysable analogues of ATP and the ATP hydrolysis transition-state analogue ADP.AlFx. Results show that different nucleotide-bound forms of PspF can change the interactions between sigma54, sigma54-RNA polymerase, and a DNA fork junction structure present within closed promoter complexes. This provides evidence that in the activation transduction pathway, several functional states of the activator, prior to ATP hydrolysis, can serve to alter the fork junction binding activity of sigma54 and sigma54-RNA polymerase that precede full DNA opening. A sequential set of nucleotide-dependent transitions in sigma54-RNA polymerase promoter complexes needed for productive open complex formation may therefore depend upon different nucleotide-bound forms of the activator.

Interactions of regulated and deregulated forms of the sigma54 holoenzyme with heteroduplex promoter DNA.

The bacterial sigma54 RNA polymerase holoenzyme binds to promoters as a stable closed complex that is silent for transcription unless acted upon by an enhancer-bound activator protein. Using DNA binding and transcription assays the ability of the enhancer-dependent sigma54 holoenzyme to interact with promoter DNA containing various regions of heteroduplex from -12 to -1 was assessed. Different DNA regions important for stabilising sigma54 holoenzyme-promoter interactions, destabilizing binding, limiting template utilisation in activator-dependent transcription and for stable binding of a deregulated form of the holoenzyme lacking sigma54 Region I were identified. It appears that homoduplex structures are required for early events in sigma54 holoenzyme promoter binding and that disruption of a repressive fork junction structure only modestly deregulates transcription. DNA opening from -5 to -1 appears important for stable engagement of the holoenzyme following activation. The regulatory Region I of sigma54 was shown to be involved in interactions with the sequences in the -5 to -1 area.