Promoters for T7 RNA polymerase have a highly conserved sequence of 23 continuous base pairs located at position -17 to +6 relative to the initiation site for the RNA.The complex of T7 RNA polymerase with the phage 410 promoter has been visualizedindirectly by exploiting the ability of the polymerase to protect DNA sequences from cleavage by methidiumpropyl-EDTA-Fe(II). The DNA contacts made by T7 RNA polymerase have been mapped during binding and during the subsequent initiation of transcription. The RNA polymerase alone protects 19 bases in a region from -21 to -3. Synthesis of the trinucleotide r(GGG) expands the length of the sequence protected by the RNA polymerase and stabilizes the complex; 29 bases (-21 to +8) are protected, and the observed equilibrium association constant of the r(GGG) complex is 5 x 105 M-1. The formation of a hexanucleotide mRNA, r(G-GGAGA), further extends the protected region; 32 bases (-21 to +11) are protected. Finally, the synthesis of a pentadecanucleotide mRNA leads to a translocation of the region protected by the protein; the sequence now protected is reduced to 24 bases (-4 to +20).Gene I ofbacteriophage T7 encodes an RNA polymerase that is responsible for the expression of the rightmost 80% of the T7 genome (1) and is also involved in the initiation of DNA replication (2-4). In contrast to the multisubunit RNA polymerases of bacteria and eukaryotes, T7 RNA polymerase consists of a single polypeptide of molecular weight 98,856 (5, 6). The physical and catalytic properties of the enzyme have been well documented (7,8).Seventeen T7 RNA polymerase promoters are present on the T7 DNA molecule (Fig. 1), and all are oriented for rightward transcription. The promoters consist of a highly conserved 23-base-pair (bp) sequence (9-11). The 410 promoter, the promoter used in this study, is one of the strongest in vivo and in vitro (9); it has a sequence identical to that of the consensus sequence derived from all 17 promoters.A

No abstract

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.

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.

Two compatible plasmids were recently reported [Ikeda et al. (1992) Nucleic Acids Res. 20, 2517-2524] that together can be used to determine whether a mutant T7 RNA polymerase promoter is active or inactive in vivo. The first plasmid, pKGP1-1, carries T7 gene 1 (the gene encoding T7 RNA polymerase) ligated to a tac promoter, while the second plasmid, pCM-X#, carries the gene encoding chloramphenicol acetyltransferase (CAT) ligated to potential T7 promoters. If the pCM-X# plasmid carries a potential T7 promoter that can be utilized by T7 RNA polymerase, then CAT is produced from transcripts generated by T7 RNA polymerase from the potential promoter on the pCM-X# plasmid. To determine whether Escherichia coli growth characteristics and chloramphenicol (cam) resistance produced by the plasmids pKGP1-1 and pCM-X# reflect the T7 promoter activity of the possible promoters carried by the pCM-X# plasmids, the in vivo and in vitro strengths of the potential T7 promoters were compared and correlated. In vivo promoter strength was determined by measuring the relative amounts of CAT present in E. coli extracts, while relative in vitro promoter strength was measured in transcription assays. The in vivo and in vitro strengths of 22 point mutants of the consensus T7 promoter were shown to correlate with the growth characteristics and cam resistance conferred to E. coli harboring the plasmid pKGP1-1 and the respective pCM-X# plasmid.(ABSTRACT TRUNCATED AT 250 WORDS)