Reiner Gentz

A T5 promoter-based transcription-translation system for the analysis of proteins in vitro and in vivo.

Publisher Summary This chapter discusses a T5 promoter-based transcription–translation system for analyzing proteins in vitro and in vivo . The chapter describes a simple method that permits the expression of cloned sequences in Escherichia coli as well as in cell-free in vitro systems of eukaryotic (wheat germ, reticulocyte, and HeLa cells) or prokaryotic ( E. coli ) origin, using a single expression unit. The expression of cloned genes in heterologous systems in vitro and in vivo has been instrumental in the identification and analysis of gene products and their derivatives. The essential element of the unit is a promoter derived from coliphage T5 that is utilized by E. coli ribonucleic acid (RNA) polymerase. It efficiently directs the synthesis of capped or uncapped mitochondrial RNA (mRNA) in vitro . Translation of such RNAs in the presence of [ 35 S] methionine yields single proteins of such high specific activity and purity that they can be directly analyzed by gel electrophoresis without the necessity of prior immunoprecipitation. The chapter also describes the application of the method for the study of structure/function relationships of proteins, including (1) protein synthesis in vivo and in vitro , (2) the translocation of proteins into and through membranes, and (3) the interruption of translation at predetermined sites for the generation and characterization of truncated proteins. Plasmid pDS5 and its derivatives 5 are members of a plasmid family developed for the study of transcriptional signals. The chapter also summarizes the essential properties of the pDS5 system.

Insertion of Transcriptional Elements Outside the Replication Region can Interfere with Replication, Maintenance, and Stability of ColE1-Derived Plasmids

The primary goal of our work is to contribute to a better understanding of structure-function relationships of nucleotide sequences defining promoters in theE. coli system. Since strong, unregulated promoters can be assumed to exhibit pertinent structural features most clearly, we investigated, in vitro, more than 50 promoters for their ability (a) to compete for the binding of RNA polymerase (an indication of their “signal strength”) and (b) to produce RNA (“overall efficiency” of a promoter) under conditions of limiting amounts of RNA polymerase. Promoters fulfilling these requirements best were found primarily in the genome of coliphage T5 (1). Some of these promoters show extremely high rates of complex formation with RNA polymerase (1,2) and at the same time were identified as being very efficient initiators of RNA synthesis in vitro and in vivo (1,3).