T7 RNA polymerase promoter

The suppressor tRNA developed by the Chamberlin lab for use in a rabbit reticulocyte lysate is based on an E. coli glycyl tRNA, which was initially chosen because glycyl-tRNA synthetases do not rely on a double-sieve editing mechanism for enzymatic hydrolysis of misacylated tRNAs [26]. Two base pair changes were made to the acceptor stem to allow incorporation of the optimal T7 RNA polymerase promoter into the DNA template for tRNA y-Con [27,28],... 

Building on earlier work of Osawa and co-workers [55], Oliver and Kowal [52] tested the feasibility of introducing a noncoded amino acid at an unassigned codon in M. luteus. DNA templates were prepared which coded for 19-mer polypeptides containing either the unassigned codon AGA(Arg) or the termination codon TAG at position 13 under the control of a T7 RNA polymerase promoter. The corresponding tRNAs, produced as described in Sect. 2, were based on tRNA and acylated with phenylalanine. The tRNA was modified to prevent recognition by the alanine aminoacyl-tRNA synthetase and to increase translational efficiency. 

Double-stranded DNA template comprising T7 RNA polymerase promoter sequence (S -ACGCACGCTGTAATACGACTCACTATA-3 promoter italicized) and reverse complement of the tagging RNA (5 -yCCCATCACCATCTCTTGCTATAGTGAGTCGTATTAC AGCGTCCGT-S boldface portion complementary to tagging RNA sequence two underlined nucleotides 2 -OMe)... 

The EMBL vector systems have been subjected to many modifications to enhance their usefulness. The most useful modifications involve the addition of more restriction enzyme recognition sites flanking the stuffer fragment and the addition of T3 and T7 RNA polymerase promoters. The T3 and T7 RNA polymerase promoters are used to synthesize 32P-labeled RNAs that can be used as probes for the identification of overlapping A clones. Many of the A vectors described above are available from or have been developed by commercial vendors. As in the case with many basic molecular biology techniques, the components needed to construct a specific recombinant phage library (A vector arms, in vitro packaging extracts, etc.) and even the library can be obtained from commercial sources. 

Rosa, M. D. (1979). Four T7 RNA Polymerase Promoters Contain an Identical 23 Base Pair Sequence. Cell 16 815. 

Tabor, S. and Richardson, C. C.(1985). A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. U. S. A. 82, 1074-1078. 

There are a number of approaches for the expression of proteins in E. coli. A modern approach to protein expression is to make use of purpose designed pDNA expression vectors such as the pET vector family, freely available from commercial sources (Figure 3.16). This pDNA expression vector family comprises a T7 RNA polymerase promoter for transcription, hence... 

Methods in which the amount of mRNA has been amplified to produce labeled cRNA, by incorporating a T7 RNA polymerase promoter site into one end of the cDNA followed by in vitro transcription are also widely used [51]. In this method, quantitative estimates of the amount of each transcript in a given sample can be calculated. In single-sample labeling experiments, a reference RNA may be spiked in during sample labeling and hybridization to facilitate quality control of the process as well as for comparison of data between different arrays. 

Radiolabelled (a32p) CTP is incorporated into multiple RNA copies using a commercial kit transcribing from a T7 RNA polymerase promoter. ... 

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