PubMed · 9108510
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A C Christensen. 1997. Bacteriophage lambda-based expression vectors.. https://doi.org/10.1385/0-89603-480-1%3A29
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Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can substantially accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in Escherichia coli based on the controlled expression of the replisome of bacteriophage T7 (T7-ORACLE). The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10-5 substitutions per base in vivo-100,000-fold above the genomic mutation rate. We demonstrated continuous evolution using the T7 replisome by expanding the substrate scope of TEM-1 β-lactamase and increasing activity 5000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than 1 week.
Three contiguous fragments of Escherichia coli 5S rRNA were prepared by T7 transcription from synthetic DNA templates. The central fragment, comprising residues 33-71 of the molecule, was transcribed in the presence of 4-thiouridine triphosphate together with [32P]UTP. The three transcripts were ligated together, yielding a 5S rRNA analogue carrying 4-thiouridine residues at positions 40, 48, 55 and 65 in helices II and III. After ligation, the 4-thiouridine residues were derivatised with p -azidophenacyl bromide. The modified 5S rRNA was reconstituted into 50S subunits and these subunits were used to prepare 70S ribosomes in the presence or absence of tRNA and mRNA. The azidophenyl groups were then photoactivated by mild irradiation at 300 nm and the products of cross-linking analysed by our standard procedures. Multiple cross-links from 5S rRNA to two distinct regions of the 23S rRNA were observed. The first region was located in helix 38 in Domain II of the 23S molecule, with cross-links at sites between nucleotides 885 and 922. The second region covered helices 81-85 in Domain V, with sites between nucleotides 2272 and 2345. Taken together with previous data, these results serve to define the arrangement of the 5S rRNA molecule relative to the 23S rRNA within the 50S subunit.
Transcription is the fundamental process by which RNA is synthesized by RNA polymerases on double-stranded DNA templates. One structurally simple RNA polymerase is encoded by bacteriophage T7. T7 RNA polymerase is an excellent candidate for studying structural aspects of transcription, because unlike the eucaryotic and bacterial RNA polymerases, it is a single subunit enzyme and does not require additional factors to carry out the entire process of transcription from start to finish. An important advantage of studying transcription using this enzyme is that the high-resolution crystal structure of T7 RNA polymerase has been solved. However, a cocrystal structure of the polymerase complexed with promoter has not yet been published. Here, we have used cross-linking techniques to understand the interaction of promoter with T7 RNA polymerase. We constructed promoters that were substituted with the photo-cross-linkable nucleotide 5-iodo uracil at every dT in the promoter from -17 to -1. This substitution replaces the 5-methyl in dT with an iodine atom. The substituted promoters were photo-cross-linked to T7 RNAP, and the efficiency of cross-linking was quantitated at every position. In the melting domain, the strongest contacts occurred at -3 and at -1 on the template strand while very weak cross-linking was seen at -2 and at -4 on the nontemplate strand. In the binding domain, the strongest contacts were seen at -16, -15, and -13 and at -10 on the template strand while at -17 and -14 on the nontemplate strand very weak cross-linking was observed. Cross-linking was poor in the intervening region between the binding and the melting domains. These results suggested that, in the T7 RNA polymerase-promoter complex, the polymerase molecule mainly contacts the template bases in the TATA box while the upstream contacts are used as an anchor for DNA binding. For a systematic study designed to probe the nature of base-specific interactions in the polymerase-promoter complex, we used neutral salts from the Hofmeister series. In general, the order of perturbation was sulfate > citrate > acetate for anions and ammonium > magnesium > potassium for cations. Using acrylamide, a neutral hydrophobic agent to probe for nonionic contacts, we observed that at -2, -4, and -17 the contacts had a hydrophobic component, while at many other positions there was no significant effect, suggesting that the contacts in the promoter-polymerase complexes were predominantly ionic but at certain positions nonionic interactions also existed. To localize a specific interaction in the melting domain, we proteolyzed the cross-linked T7 RNAP and analyzed the fragments using gel electrophoresis, mass spectrometry, and amino acid composition. High-resolution mapping indicated that amino acid residues 614-627 may be in the vicinity of the melting domain. Specifically, Y623 may contact -3 on the template strand.