Regulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo-EM.

In mammals, a distinct RNA polymerase II form, RNAPII(G) contains a novel subunit Gdown1 (encoded by POLR2M), which represses gene activation, only to be reversed by the multisubunit Mediator co-activator. Here, we employed single-particle cryo-electron microscopy (cryo-EM) to disclose the architectures of RNAPII(G), RNAPII and RNAPII in complex with the transcription initiation factor TFIIF, all to ~19 Å. Difference analysis mapped Gdown1 mostly to the RNAPII Rpb5 shelf-Rpb1 jaw, supported by antibody labelling experiments. These structural features correlate with the moderate increase in the efficiency of RNA chain elongation by RNAP II(G). In addition, our updated RNAPII-TFIIF map showed that TFIIF tethers multiple regions surrounding the DNA-binding cleft, in agreement with cross-linking and biochemical mapping. Gdown1's binding sites overlap extensively with those of TFIIF, with Gdown1 sterically excluding TFIIF from RNAPII, herein demonstrated by competition assays using size exclusion chromatography. In summary, our work establishes a structural basis for Gdown1 impeding initiation at promoters, by obstruction of TFIIF, accounting for an additional dependent role of Mediator in activated transcription.

Hybrid electron microscopy-FRET imaging localizes the dynamical C-terminus of Tfg2 in RNA polymerase II-TFIIF with nanometer precision.

TFIIF-a general transcription factor comprising two conserved subunits can associate with RNA polymerase II (RNAPII) tightly to regulate the synthesis of messenger RNA in eukaryotes. Herein, a hybrid method that combines electron microscopy (EM) and Förster resonance energy transfer (FRET) is described and used to localize the C-terminus of the second TFIIF subunit (Tfg2) in the architecture of RNAPII-TFIIF. In the first stage, a poly-histidine tag appended to the Tfg2 C-terminus was labeled with nickel-NTA nanogold and a seven-step single particle EM protocol was devised to obtain the region accessible by the nanogold in 3D, suggesting the Tfg2 C-terminus is proximal to the clamp of RNAPII. Next, the C-termini of the Rpb2 and the Rpb4 subunits of RNAPII, adjacent to the clamp, were selected for placing FRET satellites to enable the nano-positioning (NP) analysis, by which the localization precision was improved such that the Tfg2 C-terminus was found to dwell on the clamp ridge but could move to the clamp top during transcription. Because the tag receptive to the EM or FRET probes can be readily introduced to any protein subunit, this hybrid approach is generally applicable to complement cryo-EM study of many protein complexes to nanometer precision.

Does trust promote more teamwork? Modeling online game players' teamwork using team experience as a moderator.

The need for teamwork has grown significantly in today's organizations. Especially for online game communities, teamwork is an important means of online game players' engagement. This study aims to investigate the impacts of trust on players' teamwork with affective commitment and normative commitment as mediators. Furthermore, this research includes team experience as a moderator to compare the difference between different player groups. A model was proposed and tested on 296 online game players' data using structural equation modeling. Findings revealed that team experience moderated the relationship between trust and teamwork. The results indicated that trust promotes more teamwork only for players with high experience through affective commitment than those who with low experience. Implications of the findings are discussed.

Designing attractive gamification features for collaborative storytelling websites.

Gamification design is considered as the predictor of collaborative storytelling websites' success. Although aforementioned studies have mentioned a broad range of factors that may influence gamification, they neither depicted the actual design features nor relative attractiveness among them. This study aims to identify attractive gamification features for collaborative storytelling websites. We first constructed a hierarchical system structure of gamification design of collaborative storytelling websites and conducted a focus group interview with eighteen frequent users to identify 35gamification features. After that, this study determined the relative attractiveness of these gamification features by administrating an online survey to 6333 collaborative storytelling websites users. The results indicated that the top 10 most attractive gamification features could account for more than 50% of attractiveness among these 35 gamification features. The feature of unpredictable time pressure is important to website users, yet not revealed in previous relevant studies. Implications of the findings were discussed.

Development of t(50) and its application to evaluate very-high-gravity ethanol fermentation.

A three-parameter logistic growth model was modified to monitor the glucose uptake profile of yeast during very-high-gravity (VHG) ethanol fermentation. The modified model was used to define t(50) as a quantifier to differentiate among various fermentation conditions. There are two types of t(50); t(50)(g) is the time required to convert 50% of the initial glucose, and t(50)(e) is the time required to produce half of the final ethanol. A 2(4) factorial experimental design was implemented to illustrate the applicability of using t(50) to isolate active ingredients in VHG growth media. The analytical results obtained from the experimental design and from a modified model were compared, which demonstrated that t(50) could serve the proposed objectives. A shorter t(50) implies a faster fermentation. A tailing of the ethanol profile after t(50)(e) indicates that there is an inhibitory effect imposed on yeast, i.e., the stronger the tailing in the ethanol profile, the stronger the inhibitory effect. When t(50) is equal to or near to the halftime of the total course of the fermentation, a bell-shaped curve was seen for the glucose uptake rate or for the ethanol production rate, indicating that the inhibitory effect exerted on yeast was evenly distributed.