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The "B-finger" of transcription factor IIB (TFIIB) is highly conserved and believed to play a role in the initiation process. We performed alanine substitutions across the B-finger of human TFIIB, made change-of-charge mutations in selected residues, and substituted the B-finger sequence from other organisms. Mutant proteins were examined in two minimal promoter systems (containing only RNA polymerase II, TATAbinding protein, and TFIIB) and in a complex system, using TFIIB-immunodepleted HeLa cell nuclear extract (NE). Mutations in conserved residues located on the sides of the B-finger had the greatest effect on activity in both minimal promoter systems, with mutations in residues Glu-51 and Arg-66 eliminating activity. The double change-of-charge mutant (E51R: R66E) did not show activity in either minimal promoter system. Mutations in the nonconserved residues at the tip of the B-finger did not significantly affect activity. However, all of the mutations in the B-finger showed at least 25% activity in the HeLa cell NE. Chimeric proteins, containing B-finger sequences from species with conserved residues on the side of the B-finger, showed wild-type activity in a minimal promoter system and in the HeLa cell NE. However, chimeric proteins whose sequence showed divergence on the sides of the B-finger had reduced activity. Transcription factor IIF (TFIIF) partially restored activity of the inactive mutants in the minimal promoter system, suggesting that TFIIF in HeLa cell NE helps to rescue the inactive mutations by interacting with either the B-finger or another component of the initiation complex that is influenced by the B-finger.The RNA polymerase II (RNAP II) 2 initiation complex is extremely complicated and, thus, very difficult to meaningfully dissect. Genes that are transcribed into mRNA by RNAP II generally require five general transcription factors (TFs), designated TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (reviewed in Refs. 1-5). However, arguments have been made to include the "elongation" factor TFIIS (6) and the mediator complex (7) in the list of "general" transcription factors. RNAP II and most of the TFs are multimeric protein complexes. If all proteins contained in the yeast (Saccharomyces cerevisiae) RNAP II initiation complex were considered, the count of ϳ60 polypeptides would have a mass of ϳ3 MDa (8). The high resolution crystal structure of yeast RNAP II has provided invaluable insight into the topology of the RNAP II transcription initiation complex (9). Furthermore, the crystal structures of bacterial RNAP (10, 11) and RNAP from an archaeal organism (12) establish that RNAP from all three domains of life (Bacteria, Archaea, and Eukarya) show high conservation of overall structure. However, many mechanistic details of the transcription process have yet to be determined.TFIIB functions as a single polypeptide with a two-domain structure. The C-terminal domain of TFIIB (cTFIIB) contains an imperfect direct repeat motif. The co-crystal structure of cTFIIB with the DNA-bound TATA-binding protein ...

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L-Ascorbic acid (L-AsA), also known as vitamin C, is a crucial vitamin for both plants and animals due to its antioxidant properties and abilities to scavenge harmful reactive oxygen species (ROS). However, some animals such as humans lost the ability to synthesize their own L-AsA and must therefore fulfill their nutrient requirements via dietary uptake. With increasing global demand for food, studies into methods of increasing L-AsA in crops and determining how environmental stressors can affect the biosynthesis and pool size of L-AsA represent an important and growing field. Herein, quantitative enzymatic UV/Vis measurements and qualitative 13C NMR studies are used with A. thaliana plant tissues to explore biosynthesis of L-AsA. First, studies were done to adapt procedures established within the L-AsA research community for our uses. These experiments resulted in our experimental method of analyzing L-AsA in leaf tissue of leaves 6 -- 13 in 7.5-week-old A. thaliana plants of three genotypes: wt, vtc1-1, and MIOX4. Using this, the L-AsA content of A. thaliana leaves of three genotypes was investigated to provide a baseline for feeding experiments. Then, endogenous L-AsA was measured following individual leaf feeding studies of solutions of L-AsA or its precursors, L-gulono-1,4-lactone (L-GulL) and L-galactono-1,4-lactone (L-GalL), with preliminary tests being done with 13C-labeled L-AsA and L-GulL. Lastly, MIOX4 leaves were discovered to possess elevated L-AsA content beyond a certain developmental stage. This suggests developmental stage as a contributing factor to why certain groups do not see elevated content while others see significant increases.