T7 promoter contacts essential for promoter activity in vivo.

T7 RNA polymerase promoters consist of a highly conserved 23 base-pair sequence that spans the site of the initiation of transcription (+1) and extends from -17 to +6. To determine the bases within the T7 consensus promoter that are essential for promoter function a library of mutant T7 promoters was constructed, and the in vivo activity of the mutant promoters was correlated to their sequence. The library of mutant promoters was created by randomly mutagenizing the T7 phi 10 promoter between positions -22 and +6 during the synthesis of oligonucleotides containing the phi 10 promoter. The mutagenized oligonucleotides were then ligated to a promoterless chloramphenicol acetyl transferase gene creating a plasmid (pCM-X#) that can potentially express chloramphenicol acetyl transferase in the presence of T7 RNA polymerase. E. coli containing pCM-X# and a second compatible plasmid carrying T7 gene 1 (T7 RNA polymerase) were screened for chloramphenicol resistance or chloramphenicol sensitivity. The point mutations that were found to inactivate a T7 promoter are a C to A or G substitution at -7, a T to A substitution at -8, a C to A, T, or G substitution at -9, and a G to T substitution at -11.

Iron release is reduced by mutations of lysines 206 and 296 in recombinant N-terminal half-transferrin.

Human serum transferrin consists of two iron-binding lobes connected by a short peptide linker. While the high homology and structural similarity between the two halves of the molecule would suggest similar characteristics, it has been shown that the pH-dependent rate of release of iron from the N-terminal lobe is quite different from that of its C-terminal counterpart. This suggests that the N-lobe of human serum transferrin has a specific, pH-dependent, molecular mechanism for releasing iron. Sacchettini and co-workers using structural information have hypothesized that two lysines in the N-terminal lobe of ovotransferrin create a dilysine interaction and suggest that this is the trigger for pH-dependent iron release. To investigate this hypothesis, we used a Pichia pastoris expression system to produce large amounts of wild-type nTf, the single point mutants, nTfK206A (Lys 206 to alanine) and nTfK296A (Lys 296 to alanine), and the double mutant, nTfK206/296A. The purified recombinant proteins were then used to measure rates of iron release to pyrophosphate. It was found that the rate of iron release from all three mutant proteins at pH 5.7 (the pH at which nTf would normally release iron) was too slow to measure. Only when the pH was reduced to 5.0 could the rates of iron release from the mutant proteins be reliably determined. Although this precludes a direct comparison to wild-type nTf (the rate of iron release from nTf at pH 5.0 is too fast to measure), it implicates lysines 206 and 296 in the pH-dependent release of iron from nTf.