William T. McAllister

We have examined the behavior of T7 RNA polymerase (RNAP) at a set of promoter variants having all possible single base pair (bp) substitutions. The polymerase exhibits an absolute requirement for initiation with a purine and a strong preference for initiation with GTP vs ATP. Promoter variants that would require initiation at the normal start site (+1) with CTP or UTP result in a shift in initiation to +2 (with GTP). However, the choice of start site is little affected by base substitutions elsewhere in the initiation region. Furthermore, when the initiation region is shifted either one nucleotide (nt) closer or 1 nt further away from the binding region, transcription still begins the same distance downstream. These results indicate that the sequence around the start site is of little importance in start site selection and that initiation is directed a minimum distance of 5 nt downstream from the binding region. At promoters that initiate with +1 GGG, T7 RNAP synthesizes a ladder of poly(G) products as a result of slippage of the transcript on the three C residues in the template strand from +1 to +3. At promoter variants in which there is an opportunity to form a longer RNA-DNA hybrid, this G-ladder is enhanced and extended. This observation is not in agreement with recent suggestions that the RNA-DNA hybrid in the initiation complex cannot extend further than 3 bps upstream from the active site [Cheetham, G., Jeruzalmi, D., and Steitz, T. A. (1999) Nature 399, 80-83].

This paper details a facile approach for the synthesis of stable and monodisperse silver nanoparticles performed at ambient/low temperature, where Allium sativum (garlic) extract functions as the silver salt reducing agent during nanoparticle synthesis as well as the postsynthesis stabilizing ligands. Varying the synthesis conditions provides control of particle size, size‐distribution, and kinetics of particle formation. Infrared spectroscopy, energy dispersive X‐ray chemical analysis, and high‐performance liquid chromatography indicated that allicin and other carbohydrates in the garlic extract are the primary nanoparticle stabilizing moieties. The synthesized silver nanoparticles also demonstrate potential for biomedical applications, owing to (1) enhanced stability in biological media, (2) resistance to oxidation by the addition of H 2 O 2 , (3) ease and scalability of synthesis, and (4) lack of harsh chemicals required for synthesis. Cytotoxicity assays indicated no decrease in cellular proliferation for vascular smooth muscle cells and 3T3 fibroblasts at a concentration of 25 μ g/mL, confirming that silver nanoparticles synthesized with garlic extract are potential candidates for future experimentation and implementation in the biomedical field.

The high specificity of T7 RNA polymerase (RNAP) for its promoter sequence is mediated, in part, by a specificity loop (residues 742-773) that projects into the DNA binding cleft (1). Previous work demonstrated a role for the amino acid residue at position 748 (N748) in this loop in discrimination of the base pairs (bp) at positions -10 and -11 (2). A comparison of the sequences of other phage RNAPs and their promoters suggested additional contacts that might be important in promoter recognition. We have found that changing the amino acid residue at position 758 in T7 RNAP results in an enzyme with altered specificity for the bp at position -8. The identification of two amino acid:base pair contacts (i.e., N748 with the bp at -10 and -11, and Q758 with the bp at -8) provides information concerning the disposition of the specificity loop relative to the upstream region of the promoter. The results suggest that substantial rearrangements of the loop (and/or the DNA) are likely to be required to allow these amino acids to interact with their cognate base pairs during promoter recognition.