The use of rifampicin to evaluate tRNA transcriptional organization in Escherichia coli

Abstract

The antibiotic rifampicin, which in prokaryotes inhibits the initiation of RNA synthesis but not the completion of nascent strands, was used to explore tRNA gene transcriptional organization in Escherichia coli. Cultures were grown in [32P] orthophosphate to constant specific radioactivity and labeled with [3H] uridine in the presence of rifampicin. Numerous tRNA species then were isolated by polyacrylamide gel electrophoresis and their 3H/32P ratios determined; these ratios, following correction for the base compositions of the tRNAs, should reflect the distances of the corresponding tRNA genes from their promoters. Individual tRNA species were identified, where possible, by oligonucleotide fingerprint analysis. Observed isotopic ratios were correlated with promoter-gene distances, measured in nucleotides, using the nucleotide sequence of the 16S ribosomal RNA gene as a reference. The protocols developed should be applicable to most prokaryotes.