A coupled DNA-directed in vitro system to study gene expression based on di- and tripeptide formation.
In this report, a simplified coupled DNA-directed in vitro system has been described that is based on the formation of the first di- or tripeptide of the gene product. This system is gene specific and quantitative, and the assay (especially the extraction procedure) is very rapid. The fact that both transcription and translation initiation occur in this system makes it ideally suited for studies on the regulation of prokaryotic gene expression. The ideal templates are plasmids, DNA fragments or purified mRNAs that direct the synthesis of a limited number of products with different second amino acids. An essential requirement is that the initial sequence of the protein products be known, although this system could be used to determine the second amino acid in cases where there is some doubt from the DNA sequence as to where a particular protein initiates. A difficulty arises when a plasmid contains more than one gene whose protein products have the same initial dipeptide. One solution to the problem is to measure tripeptide formation if the third amino acid is different. A second procedure, if the code word for the second amino acid differs between the genes, is to use purified isoacceptor tRNA species to distinguish the products. Another important application of tripeptide synthesis is that it can be used as a measure of the amount of active mRNA present in a mixture of mRNAs. The use of a ribosomal high-salt wash instead of the purified initiation and elongation factors greatly simplifies this system and should make it suitable for routine analysis in most laboratories.
D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D004151:Dipeptides / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005818:Genetic Engineering / Q000379:methods; D007700:Kinetics; D009842:Oligopeptides / Q000235:genetics*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics
D004269:DNA, Bacterial; D004151:Dipeptides; D009842:Oligopeptides; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; D004247:DNA; D004262:DNA Restriction Enzymes