Heterogeneous initiation due to slippage at the bacteriophage 82 late gene promoter in vitro

RNAs synthesized in vitro by purified Escherichia coli RNA polymerase from a bacteriophage 82 promoter are heterogeneous at the 5' end. We show that this heterogeneity results from variable addition of extra adenine residues, allowed by slippage of the initial oligonucleotide pppAAA-OH against its DNA template sequence TTT. Slippage backward by one base allows another A to be added, giving pppAAAA-OH, and this cycle can continue more than 20 times before it is ended by incorporation of UMP encoded by the fourth template base A. Slippage is abolished by mutation of the TTT template sequence to TGT and is sensitive to the concentrations of UTP and ATP in the reaction mixture. Analysis of deletions, substitutions, and point mutants implies that the slippage reaction requires only the existence of TTT at the initiation site of the template strand, although changes in neighboring nucleotides slightly affect its efficiency.