Pseudo-templated transcription by Escherichia coli RNA polymerase at a mutant promoter

Abstract

A G → T mutation at the start-point of transcription of the phage P22 sar promoter (sar+ IT] causes a novel defect in promoter clearance by Escherichia coli RNA polymerase (RNAP] in vitro. Under standard transcription conditions, in the presence of high concentrations of all four NTPs, the predominant products from this promoter are poly(U) chains of varying length. Because the mutation creates a run of four T : A base-pairs from - 1 to + 3 (TGTT → TTTT), we propose that synthesis of poly(U) is pseudo-templated by the A4 stretch on the template strand. G → A and G → C mutations at position 4-1 do not cause pseudo-templated transcription. Several molecules of poly(U) are produced and released per sar+1T promoter-polymerase complex without dissociation of RNAP from the template DNA. The exponential relationship between yield and size of individual poly(U) species indicates that there is a constant probability that another U residue will be added to the nascent chain. Presumably, pseudo-templated transcription occurs by a slippage (stuttering) mechanism like that proposed to explain certain kinds of RNA editing in eukaryotic viral mRNAs.